Recent Advances in the Treatment of Faecal Incontinence

*Current Topics in Faecal Incontinence*

[36] Braham A, Safer L, Bdioui F, et al. Antiphospholipid antibodies in digestive diseases. Presse Médicale.

[37] Asherson RA, Cervera R. Unusual manifestations of the antiphospholipid syndrome. Clinical Reviews in Allergy and Immunology. 2003;**25**:61-78

antiphospholipid syndrome as a new cause of autoimmune pancreatitis. Gut.

[39] Hannu T, Mattilla L, Siitonen A, et al. Reactive arthritis attributable to Shigella infection: A clinical and epidemiological nationwide study. Annals of the Rheumatic Diseases.

[40] Orlando A, Renna S, Perricone G, et al. Gastrointestinal lesions associated with spondyloarthopaties. World Journal of Gastroenterology.

[41] Rudwaleit M, Baeten D. Ankylosing

spondylitis and bowel disease. Best Practice & Research. Clinical Rheumatology. 2006;**20**:451-466

[42] Smale S, Natt RS, Orchard TR. Inflammatory bowel disease and spondylarthropathy. Arthritis and Rheumatism. 2001;**44**:2728-2736

[43] De Keyser F, Baeten D, Van den Bosch F, et al. Gut inflammation and spondyloarthropathies. Current Rheumatology Reports. 2002;**4**:525-532

[44] Yurdakul S, Tuzuner N, Yardakul I, et al. Gastrointestinal involvement in Behçet's syndrome: A controlled study. Annals of the Rheumatic Diseases.

2001;**30**:1890-1897

[38] Spencer HL. Primary

2004;**53**:468

2005;**64**:594-598

2009;**15**:2443-2448

**78**

1996;**55**:208-210

**81**

**Chapter 6**

**Abstract**

Incontinence

*John Camilleri-Brennan*

results of the various studies to date.

**1. Introduction**

anal bulking agents, gatekeeper, Sphinkeeper

Anal Injectable and Implantable

Faecal incontinence (FI) is a common condition, the prevalence of which increases with age. It is associated with a negative impact on the quality of one's life. The aetiology is multifactorial; hence, both the diagnosis and the treatment of faecal incontinence may be challenging. A variety of surgical treatments for faecal incontinence have emerged over the years. One of these is the use of anal bulking agents. Anal bulking agents have been available for over 25 years, with various studies being published. Initial results were disappointing, mainly due to lack of efficacy and reliability as well as concerns about safety. Great strides have been made recently with the introduction of the anal implants Gatekeeper (GK) and Sphinkeeper (SK). This chapter explores the evolution of anal injectables and implants, discusses operative techniques and provides a critical analysis of the

**Keywords:** faecal incontinence, anal sphincter, anal implants, anal injectables,

Faecal incontinence (FI) may be defined as an impaired ability of the control of the release of flatus or faeces. It is a socially stigmatising condition that may have an adverse effect on one's quality of life. From the financial point of view, the investigation and treatment of faecal incontinence may add to a significant cost to the health systems of most countries. In fact, the annual treatment cost of patients

Many factors may be involved in the pathophysiology of FI. A thorough clinical assessment of the patient is therefore mandatory. This starts with a full history, which may include a cognitive assessment if necessary. The characteristics of the faeces and the type and frequency of incontinence should be noted. Urge incontinence is suggestive of poor external anal sphincter (EAS) function, whilst passive and post-defaecatory incontinence indicates that internal anal sphincter (IAS) function is weak. Various questionnaires that enable the clinician to quantify the degree of incontinence

in the UK with urinary and faecal incontinence is of about £500 million.

and the impact on quality of life are available. These include symptom-specific questionnaires, such as the ones developed by Vaizey et al. [1] and Wexner et al. [2]

Bulking Agents for Faecal

#### **Chapter 6**

## Anal Injectable and Implantable Bulking Agents for Faecal Incontinence

*John Camilleri-Brennan*

#### **Abstract**

Faecal incontinence (FI) is a common condition, the prevalence of which increases with age. It is associated with a negative impact on the quality of one's life. The aetiology is multifactorial; hence, both the diagnosis and the treatment of faecal incontinence may be challenging. A variety of surgical treatments for faecal incontinence have emerged over the years. One of these is the use of anal bulking agents. Anal bulking agents have been available for over 25 years, with various studies being published. Initial results were disappointing, mainly due to lack of efficacy and reliability as well as concerns about safety. Great strides have been made recently with the introduction of the anal implants Gatekeeper (GK) and Sphinkeeper (SK). This chapter explores the evolution of anal injectables and implants, discusses operative techniques and provides a critical analysis of the results of the various studies to date.

**Keywords:** faecal incontinence, anal sphincter, anal implants, anal injectables, anal bulking agents, gatekeeper, Sphinkeeper

#### **1. Introduction**

Faecal incontinence (FI) may be defined as an impaired ability of the control of the release of flatus or faeces. It is a socially stigmatising condition that may have an adverse effect on one's quality of life. From the financial point of view, the investigation and treatment of faecal incontinence may add to a significant cost to the health systems of most countries. In fact, the annual treatment cost of patients in the UK with urinary and faecal incontinence is of about £500 million.

Many factors may be involved in the pathophysiology of FI. A thorough clinical assessment of the patient is therefore mandatory. This starts with a full history, which may include a cognitive assessment if necessary. The characteristics of the faeces and the type and frequency of incontinence should be noted. Urge incontinence is suggestive of poor external anal sphincter (EAS) function, whilst passive and post-defaecatory incontinence indicates that internal anal sphincter (IAS) function is weak. Various questionnaires that enable the clinician to quantify the degree of incontinence and the impact on quality of life are available. These include symptom-specific questionnaires, such as the ones developed by Vaizey et al. [1] and Wexner et al. [2]

#### *Current Topics in Faecal Incontinence*

and the faecal incontinence quality of life (FIQOL) scale developed by Rockwood et al. [3], and also generic questionnaires such as the Short Form 36 (SF 36) [4].

A full examination of the patient, including the abdomen and perineum and a neurological examination in some cases, is necessary. Beneficial investigations include a flexible sigmoidoscopy, anal manometry (resting and squeeze pressure), rectal compliance, pudendal nerve terminal motor latency (PNTML) and endoanal ultrasound (EAUS). Clinicians, however, need to be able to determine which test to perform, and when, as well as be able to correctly interpret the results.

The management of FI is complex and multidisciplinary, involving the general practitioner, continence nurse, physiotherapist, gastroenterologist, urologist and colorectal surgeon. Conservative measures, which include patient education and support, improvement in diet and bowel habit, judicious use of anti-diarrhoeal medication and pelvic floor exercises, are used in the first instance. This is, in fact, recommended in the UK by the National Institute for Clinical Excellence (NICE) guideline 'CG49 Faecal Incontinence' [5]. If these measures fail, surgical intervention may be necessary. A variety of surgical options are available, with the appropriate therapy being selected depending on the cause of the incontinence and the patient's cognitive function and general physical condition (**Table 1**). One of the surgical options available is the use of anal bulking agents.

#### 1.**Restoration and improvement of residual sphincter function**

a. Correcting a defective external anal sphincter

Sphincteroplasty (end-to-end repair; overlap repair)

b. Correcting a defective pelvic floor:

Levatorplasty

Postanal repair


#### 2.**Increasing the outlet resistance of the anal sphincter**

a. Augmentation of the anal sphincter and anal cushions (anal bulking agents)

**83**

**2.3 The second phase**

have been used in these studies (**Table 2**).

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

Anal bulking agents have emerged as a treatment for FI, following the success of bulking agents for urinary stress incontinence in females. In the urology setting, bulking agents have been employed to augment the bladder neck and increase urethral resistance [6]. Therefore, the aim of anal bulking agents is to prevent FI by

The ideal characteristics of a bulking agent have been described in the literature [7]. The injected or implanted substance should be biocompatible, non-migratory, non-allergenic and noncarcinogenic. The substance should also be easy to inject or implant and should produce an improvement in continence, both in the short term

Anal injectables and implantables have been used to manage faecal incontinence for over 20 years. It may be useful to chart their development over the years and to classify this development into three phases. The first phase consists of the initial experimental studies that took place in the 1990s. The second phase, from about the year 2000 onwards, encompasses an increase in the number of studies using a wide variety of agents and injection techniques. The third phase features the latest generation of anal bulking agents, the implantable polyacrylonitrile, available as

Anal bulking agents were first described in 1993 by Shafik [8]. Shafik, an Egyptian surgeon, is considered to be a pioneer in this field. In his first study, he described the outcomes following the injection of 5 ml of PTFE (polytef/Teflon) paste in 11 patients, 7 of whom had incontinence following a lateral internal sphincterotomy for anal fissure. In another study, the same author used 60 ml of abdominal wall fat as a submucosal injection into the rectal neck at 3 and 9 o'clock in 14 patients with partial faecal incontinence [9]. Pescatori's group from Rome, Italy, reported the use of anal injection of autologous buttock fat to restore continence in one patient who had poor results following a sphincteroplasty. This patient's

The indications for injection of the anal bulking agents in these studies were various. Most patients had passive FI, but some had urge incontinence, indicating EAS disruption. The results of these initial studies showed that continence was improved in the short term. However, the medium- and long-term results were poor, probably because of the resorption or migration of the injected material.

A number of safety issues were raised with these studies. Teflon could potentially cause granuloma formation and sarcomas. The injection of autologous fat as a bulking agent in urology has been implicated in fatal fat embolism and stroke.

The second phase in the development of anal bulking agents consisted of a wide variation in the types of materials used, surgical technique and clinical indications [11]. Some of the materials used to bulk the anal sphincter were being used in urology to augment the bladder neck. Nine different types of injectable bulking agents

closing the anal canal or increasing the pressure within the anal sphincter.

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

**2.1 The evidence for anal bulking agents**

Gatekeeper (GK) and Sphinkeeper (SK) devices.

continence improved following repeated injections [10].

Reinjection was necessary in order to maintain continence.

**2.2 Initial studies: The first phase**

**2. Anal bulking agents**

and in the long term.


#### 3.**Dynamic sphincter replacement**


#### 4.**Antegrade continence enema (ACE)**

	- a. Colostomy
	- b. Ileostomy

#### **Table 1.**

*Surgical options in the management of faecal incontinence.*

#### **2. Anal bulking agents**

*Current Topics in Faecal Incontinence*

and the faecal incontinence quality of life (FIQOL) scale developed by Rockwood et al. [3], and also generic questionnaires such as the Short Form 36 (SF 36) [4]. A full examination of the patient, including the abdomen and perineum and a neurological examination in some cases, is necessary. Beneficial investigations include a flexible sigmoidoscopy, anal manometry (resting and squeeze pressure), rectal compliance, pudendal nerve terminal motor latency (PNTML) and endoanal ultrasound (EAUS). Clinicians, however, need to be able to determine which test to

The management of FI is complex and multidisciplinary, involving the general practitioner, continence nurse, physiotherapist, gastroenterologist, urologist and colorectal surgeon. Conservative measures, which include patient education and support, improvement in diet and bowel habit, judicious use of anti-diarrhoeal medication and pelvic floor exercises, are used in the first instance. This is, in fact, recommended in the UK by the National Institute for Clinical Excellence (NICE) guideline 'CG49 Faecal Incontinence' [5]. If these measures fail, surgical intervention may be necessary. A variety of surgical options are available, with the appropriate therapy being selected depending on the cause of the incontinence and the patient's cognitive function and general physical condition (**Table 1**). One of the

perform, and when, as well as be able to correctly interpret the results.

surgical options available is the use of anal bulking agents.

1.**Restoration and improvement of residual sphincter function**

Sphincteroplasty (end-to-end repair; overlap repair)

a. Correcting a defective external anal sphincter

b. Correcting a defective pelvic floor:

Total pelvic floor repair c. Correction of anorectal deformities d. Sacral nerve stimulation (SNS)

e. Posterior tibial nerve stimulation (PTNS)

b. Anal submucosal fibrosis (SECCA) c. Anal encirclement (Thiersch procedure)

4.**Antegrade continence enema (ACE)**

*Surgical options in the management of faecal incontinence.*

d. Non-dynamic graciloplasty 3.**Dynamic sphincter replacement**

a. Dynamic graciloplasty b. Artificial anal sphincter

5.**Faecal diversion** a. Colostomy b. Ileostomy

2.**Increasing the outlet resistance of the anal sphincter**

a. Augmentation of the anal sphincter and anal cushions (anal bulking agents)

Levatorplasty Postanal repair

**82**

**Table 1.**

Anal bulking agents have emerged as a treatment for FI, following the success of bulking agents for urinary stress incontinence in females. In the urology setting, bulking agents have been employed to augment the bladder neck and increase urethral resistance [6]. Therefore, the aim of anal bulking agents is to prevent FI by closing the anal canal or increasing the pressure within the anal sphincter.

The ideal characteristics of a bulking agent have been described in the literature [7]. The injected or implanted substance should be biocompatible, non-migratory, non-allergenic and noncarcinogenic. The substance should also be easy to inject or implant and should produce an improvement in continence, both in the short term and in the long term.

#### **2.1 The evidence for anal bulking agents**

Anal injectables and implantables have been used to manage faecal incontinence for over 20 years. It may be useful to chart their development over the years and to classify this development into three phases. The first phase consists of the initial experimental studies that took place in the 1990s. The second phase, from about the year 2000 onwards, encompasses an increase in the number of studies using a wide variety of agents and injection techniques. The third phase features the latest generation of anal bulking agents, the implantable polyacrylonitrile, available as Gatekeeper (GK) and Sphinkeeper (SK) devices.

#### **2.2 Initial studies: The first phase**

Anal bulking agents were first described in 1993 by Shafik [8]. Shafik, an Egyptian surgeon, is considered to be a pioneer in this field. In his first study, he described the outcomes following the injection of 5 ml of PTFE (polytef/Teflon) paste in 11 patients, 7 of whom had incontinence following a lateral internal sphincterotomy for anal fissure. In another study, the same author used 60 ml of abdominal wall fat as a submucosal injection into the rectal neck at 3 and 9 o'clock in 14 patients with partial faecal incontinence [9]. Pescatori's group from Rome, Italy, reported the use of anal injection of autologous buttock fat to restore continence in one patient who had poor results following a sphincteroplasty. This patient's continence improved following repeated injections [10].

The indications for injection of the anal bulking agents in these studies were various. Most patients had passive FI, but some had urge incontinence, indicating EAS disruption. The results of these initial studies showed that continence was improved in the short term. However, the medium- and long-term results were poor, probably because of the resorption or migration of the injected material. Reinjection was necessary in order to maintain continence.

A number of safety issues were raised with these studies. Teflon could potentially cause granuloma formation and sarcomas. The injection of autologous fat as a bulking agent in urology has been implicated in fatal fat embolism and stroke.

#### **2.3 The second phase**

The second phase in the development of anal bulking agents consisted of a wide variation in the types of materials used, surgical technique and clinical indications [11]. Some of the materials used to bulk the anal sphincter were being used in urology to augment the bladder neck. Nine different types of injectable bulking agents have been used in these studies (**Table 2**).


**85**

**Figure 1.**

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

• Failure of conservative management of faecal incontinence.

The clinical indications for which these bulking agents were used varied from

• Structurally intact but weak internal anal sphincter. This would be due to either primary idiopathic degeneration of the IAS or degeneration secondary

• IAS damage (childbirth, haemorrhoidectomy, anal stretch, sphincterotomy)

The main indication was IAS dysfunction or disruption. Unlike the EAS, the IAS

The bulking agents may be inserted under local, regional (anal or pudendal nerve block) or general anaesthesia. The type of anaesthesia used depends on the preference of the patient and the surgeon. The patient may be positioned in the prone (jackknife), lithotomy or left lateral positions, although the latter position may not give a satisfactory view of the anorectum to enable accurate injection. A phosphate enema is usually administered preoperatively. The procedure is usually covered by prophylactic antibiotics, such as intravenous (IV) co-amoxiclav 1.2 g, cefuroxime 750 mg and metronidazole 500 mg or gentamicin 1.5 mg/kg and

*Endoanal ultrasound scan showing a defect in the IAS of a 57-year-old lady with passive faecal incontinence following haemorrhoidectomy. The defect is present between the arrows from the 3 to the 5 o'clock positions.*

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

to tissue disorders such as scleroderma.

• Defect in the external anal sphincter.

is not amenable to surgical repair.

*2.3.2 Surgical procedure and technique*

metronidazole 500 mg at induction.

*2.3.1 Indications*

study to study. These were:

(**Figure 1**).

**Table 2.**

*Injectable materials used in the second phase of studies.*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

#### *2.3.1 Indications*

*Current Topics in Faecal Incontinence*

**Commercial name(s)**

PTQ; Bioplastique

NASHA Dx, Zuidex, Solesta

Permacol Submucosal;

intersphincteric

Intersphincteric; within IAS

Durasphere Submucosal Transmucosal;

**Injection site Injection route Published** 

transsphincteric

Submucosal Transmucosal 5 192

Coaptite Submucosal Transsphincteric 1 10

Contigen Submucosal Transmucosal 2 90

Bulkamid Intersphincteric Intersphincteric 1 5

Onyx34 Intersphincteric Intersphincteric 1 21

Transmucosal; intersphincteric

Submucosal Transmucosal 1 6

**studies**

Transsphincteric 21 619

**No. of patients**

7 187

5 172

**Type of bulking** 

Carbon-coated zirconium beads, comprised of pyrolytic carbon-coated beads suspended in a water-based carrier gel containing β-glucan

Spherical particles of calcium hydroxylapatite, suspended in a gel

Dextranomer microspheres and stabilised sodium hyaluronate in phosphate-buffered 0.9% sodium chloride

solution

(2.5%)

fluid

Glutaraldehyde crosslinked collagen

Cross-linked porcine dermal collagen matrix

8% ethylene vinyl alcohol copolymer dissolved in dimethyl sulfoxide. A spongy solid mass forms from the solidification of the hydrophobic copolymer when the solvent diffuses away on contact with tissue

Expandable silicone microballoons filled with a biocompatible hydrogel made of poly-N-vinyl-pyrrolidone

*Injectable materials used in the second phase of studies.*

Synthetic nonparticulate hydrogel consisting of water (97.5%) and crosslinked polyacrylamide

Silicone biomaterial. Polydimethylsiloxane elastomer particles suspended in a biocompatible hydrogel made of poly-N-vinyl-pyrrolidone

**agent**

**84**

**Table 2.**

The clinical indications for which these bulking agents were used varied from study to study. These were:


The main indication was IAS dysfunction or disruption. Unlike the EAS, the IAS is not amenable to surgical repair.

#### *2.3.2 Surgical procedure and technique*

The bulking agents may be inserted under local, regional (anal or pudendal nerve block) or general anaesthesia. The type of anaesthesia used depends on the preference of the patient and the surgeon. The patient may be positioned in the prone (jackknife), lithotomy or left lateral positions, although the latter position may not give a satisfactory view of the anorectum to enable accurate injection. A phosphate enema is usually administered preoperatively. The procedure is usually covered by prophylactic antibiotics, such as intravenous (IV) co-amoxiclav 1.2 g, cefuroxime 750 mg and metronidazole 500 mg or gentamicin 1.5 mg/kg and metronidazole 500 mg at induction.

#### **Figure 1.**

*Endoanal ultrasound scan showing a defect in the IAS of a 57-year-old lady with passive faecal incontinence following haemorrhoidectomy. The defect is present between the arrows from the 3 to the 5 o'clock positions.*

The injection of the bulking agent varies depending on the type of substance used and the clinical indications. Three different routes of needle insertion were mentioned in the literature: transmucosal, transsphincteric or intersphincteric. The bulking agent was placed submucosally, within the intersphincteric space or within the IAS itself. For example, porcine dermal collagen (Permacol) may be injected via the transmucosal or transsphincteric route using a disposable 19G needle [12] (**Figure 2**). In patients with an intact IAS, 2.5 ml of Permacol is equally injected into the submucosal space at the 3, 7 and 11 o'clock positions above the dentate line. In cases of an IAS defect, 5 ml of Permacol may be injected at the site of the defect, with 2.5 ml of the substance injected diametrically opposite. With silicone biomaterial (PTQ or Bioplastique), four doses of 2.5 ml of silicone are used, using an 18G needle [13, 14]. Patients with an intact IAS have the silicone injected transsphincterically into the intersphincteric space at the 2, 4, 8 and 10 o'clock positions. In patients with an IAS defect, for example, after a lateral internal sphincterotomy, a total of three doses of 2.5 ml of silicone are injected into the defect. A fourth dose is injected into the intersphincteric space contralateral to the IAS defect, to provide symmetry. With carbon-coated beads (Durasphere), a total of 10 ml is injected in four divided doses in the submucosal plane using an 18G needle [14].

It is of utmost importance to ensure that the anal mucosa is not breached during injection, since that would allow intra-anal leakage of the substance. Intravascular injection must also be avoided.

Once the injection is completed, it is a good practice to leave the needle and syringe in place for a few seconds. As the needle is being withdrawn, pressure on the needle track by the index finger may prevent leakage of the bulking agent [12].

The bulking agent may be injected freehand, with an anal retractor such as Eisenhammer used to identify the IAS and intersphincteric groove. A finger placed within the anal canal may be useful to guide the needle to its correct position. However endoanal ultrasound has been recommended to guide the needle to an optimum position [13], especially if the agent is to be injected into the intersphincteric space or adjacent to a defect in the IAS.

#### *2.3.3 Results*

The majority of studies in this second phase of development were mainly case series and observational studies. Most of these studies reported either an improvement in the faecal continence scores or less frequent episodes of incontinence over time. Anorectal manometry testing is featured in some studies, with some showing an improvement in resting or squeeze pressures. Others studies showed no such improvement. Clinical improvement was not always associated with an increase in

**87**

disease.

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

these pressures. Quality of life was formally assessed in some of these studies. The majority reported an improvement across various domains such as physical and

To date there have been 6 randomised trials using anal bulking agents, with more than 400 patients. Two trials compared a bulking agent with a sham or saline injection. Siproudhis et al. in 2007 [15] compared a silicone biomaterial (PTQ ) with a normal saline injection (control) into the intersphincteric space. PTQ did not demonstrate any appreciable clinical benefit when compared to the control. The trial was however deemed to be too small to detect any differences in continence. Graf et al. in 2011 [16] compared the injection of dextranomer (NASHA Dx) against sham injection (no substance injected). Continence was better in the short term (6 months) in the active intervention group, although interestingly about 30% of patients in the control group had an improvement in their continence. This same group, the NASHA Dx study group, published the results of a prospective multicentre trial in 2014, showing that 'submucosal injection of NASHA Dx provided a significant improvement of FI symptoms in a majority of patients and this effect was stable during the course of the follow-up and

A small study with 10 patients by Maeda et al. in 2008 [17] revealed significant improvement at 6 weeks postinjection using injection of Bulkamid and Permacol. Continence decreased slightly in the Permacol group at 6 months. However there was no reported difference between the two agents. The numbers were too small to detect a difference. Tjandra et al. in 2009 reported the results of a randomised study comparing PTQ with carbon-coated beads (Durasphere) [14]. PTQ injection was associated with better continence scores and quality of life and was safer than

Tjandra et al. in 2004 reported the short-term benefits from ultrasound-guided

The follow-up for the majority of patients in studies was less than a median of 3 years. A question on the term durability and effectiveness of these agents is therefore raised. The majority (97%) of patients were only followed up once or twice. No long-term evidence on outcomes was available, and further conclusions were not warranted from the available data. None of the studies reported patient evaluation of outcomes, and thus it is difficult to gauge whether the improvement in the continence scores matched the practical symptom and quality of life improvements

The majority of patients did not report any complications. The complications described were mainly pain, anal bruising and leakage of injected material [11, 12]. Less common complications were anal ulceration and infection (local cellulitis and abscess formation). There were two reported cases of local giant cell foreign body reaction after injection of silicone (PTQ ) [18]. Durasphere has been associated with skin rashes and arthritis. Skin patch testing is therefore recommended before using

A relatively new and innovative development in anal bulking technology is the Gatekeeper and Sphinkeeper (THD S.p.A., Correggio, Italy). The material used is polyacrylonitrile (Hyexpan). Polyacrylonitrile is an inert, non-allergenic, nondegradable material that is also non-immunogenic and noncarcinogenic. First developed by Medtronic in Minneapolis, USA, it was originally used as an implant in the oesophagogastric junction for the management of gastro-oesophageal reflux

**2.4 The third phase: The implantable gatekeeper and Sphinkeeper**

injection of silicone biomaterial (PTQ ) compared with digital guidance [13].

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

social function.

maintained for 3 years'.

that mattered to the patients.

Durasphere.

this agent [14].

**Figure 2.** *Porcine dermal collagen (Permacol) in a 2.5 ml syringe.*

#### *Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

*Current Topics in Faecal Incontinence*

injection must also be avoided.

*2.3.3 Results*

teric space or adjacent to a defect in the IAS.

*Porcine dermal collagen (Permacol) in a 2.5 ml syringe.*

The injection of the bulking agent varies depending on the type of substance used and the clinical indications. Three different routes of needle insertion were mentioned in the literature: transmucosal, transsphincteric or intersphincteric. The bulking agent was placed submucosally, within the intersphincteric space or within the IAS itself. For example, porcine dermal collagen (Permacol) may be injected via the transmucosal or transsphincteric route using a disposable 19G needle [12] (**Figure 2**). In patients with an intact IAS, 2.5 ml of Permacol is equally injected into the submucosal space at the 3, 7 and 11 o'clock positions above the dentate line. In cases of an IAS defect, 5 ml of Permacol may be injected at the site of the defect, with 2.5 ml of the substance injected diametrically opposite. With silicone biomaterial (PTQ or Bioplastique), four doses of 2.5 ml of silicone are used, using an 18G needle [13, 14]. Patients with an intact IAS have the silicone injected transsphincterically into the intersphincteric space at the 2, 4, 8 and 10 o'clock positions. In patients with an IAS defect, for example, after a lateral internal sphincterotomy, a total of three doses of 2.5 ml of silicone are injected into the defect. A fourth dose is injected into the intersphincteric space contralateral to the IAS defect, to provide symmetry. With carbon-coated beads (Durasphere), a total of 10 ml is injected in

four divided doses in the submucosal plane using an 18G needle [14].

It is of utmost importance to ensure that the anal mucosa is not breached during injection, since that would allow intra-anal leakage of the substance. Intravascular

Once the injection is completed, it is a good practice to leave the needle and syringe in place for a few seconds. As the needle is being withdrawn, pressure on the needle track by the index finger may prevent leakage of the bulking agent [12]. The bulking agent may be injected freehand, with an anal retractor such as Eisenhammer used to identify the IAS and intersphincteric groove. A finger placed within the anal canal may be useful to guide the needle to its correct position. However endoanal ultrasound has been recommended to guide the needle to an optimum position [13], especially if the agent is to be injected into the intersphinc-

The majority of studies in this second phase of development were mainly case series and observational studies. Most of these studies reported either an improvement in the faecal continence scores or less frequent episodes of incontinence over time. Anorectal manometry testing is featured in some studies, with some showing an improvement in resting or squeeze pressures. Others studies showed no such improvement. Clinical improvement was not always associated with an increase in

**86**

**Figure 2.**

these pressures. Quality of life was formally assessed in some of these studies. The majority reported an improvement across various domains such as physical and social function.

To date there have been 6 randomised trials using anal bulking agents, with more than 400 patients. Two trials compared a bulking agent with a sham or saline injection. Siproudhis et al. in 2007 [15] compared a silicone biomaterial (PTQ ) with a normal saline injection (control) into the intersphincteric space. PTQ did not demonstrate any appreciable clinical benefit when compared to the control. The trial was however deemed to be too small to detect any differences in continence. Graf et al. in 2011 [16] compared the injection of dextranomer (NASHA Dx) against sham injection (no substance injected). Continence was better in the short term (6 months) in the active intervention group, although interestingly about 30% of patients in the control group had an improvement in their continence. This same group, the NASHA Dx study group, published the results of a prospective multicentre trial in 2014, showing that 'submucosal injection of NASHA Dx provided a significant improvement of FI symptoms in a majority of patients and this effect was stable during the course of the follow-up and maintained for 3 years'.

A small study with 10 patients by Maeda et al. in 2008 [17] revealed significant improvement at 6 weeks postinjection using injection of Bulkamid and Permacol. Continence decreased slightly in the Permacol group at 6 months. However there was no reported difference between the two agents. The numbers were too small to detect a difference. Tjandra et al. in 2009 reported the results of a randomised study comparing PTQ with carbon-coated beads (Durasphere) [14]. PTQ injection was associated with better continence scores and quality of life and was safer than Durasphere.

Tjandra et al. in 2004 reported the short-term benefits from ultrasound-guided injection of silicone biomaterial (PTQ ) compared with digital guidance [13].

The follow-up for the majority of patients in studies was less than a median of 3 years. A question on the term durability and effectiveness of these agents is therefore raised. The majority (97%) of patients were only followed up once or twice. No long-term evidence on outcomes was available, and further conclusions were not warranted from the available data. None of the studies reported patient evaluation of outcomes, and thus it is difficult to gauge whether the improvement in the continence scores matched the practical symptom and quality of life improvements that mattered to the patients.

The majority of patients did not report any complications. The complications described were mainly pain, anal bruising and leakage of injected material [11, 12]. Less common complications were anal ulceration and infection (local cellulitis and abscess formation). There were two reported cases of local giant cell foreign body reaction after injection of silicone (PTQ ) [18]. Durasphere has been associated with skin rashes and arthritis. Skin patch testing is therefore recommended before using this agent [14].

#### **2.4 The third phase: The implantable gatekeeper and Sphinkeeper**

A relatively new and innovative development in anal bulking technology is the Gatekeeper and Sphinkeeper (THD S.p.A., Correggio, Italy). The material used is polyacrylonitrile (Hyexpan). Polyacrylonitrile is an inert, non-allergenic, nondegradable material that is also non-immunogenic and noncarcinogenic. First developed by Medtronic in Minneapolis, USA, it was originally used as an implant in the oesophagogastric junction for the management of gastro-oesophageal reflux disease.

#### *Current Topics in Faecal Incontinence*

The main indications for the use of the GK and SK are passive faecal incontinence, secondary to IAS dysfunction or damage, where conservative measures or injection of other bulking agents such as PTQ or Permacol has failed. However, the use of GK and SK in patients with other causes of FI is being explored.

The following are contraindications to the use of the GK and SK. Similar contraindications have also been described by the product manufacturers of other anal bulking agents:


#### *2.4.1 Surgical apparatus, procedure and technique*

Whereas the anal bulking agents that were developed in phases 1 and 2 are injected into or around the anal canal by means of a hypodermic syringe, the Hyexpan prostheses are implanted into the intersphincteric space using a custommade gun (**Figure 3**).

The difference between GK and SK lies in the size of the prostheses. The dehydrated GK prostheses consist of thin solid cylinders, 22 mm long and 2 mm in diameter. The success of this material depends on its hydrophilic properties.

#### **Figure 3.**

*The gatekeeper gun, made of the dispenser that houses one prosthesis and the delivery system. The Sphinkeeper delivery system and dispenser are similar but slightly larger.*

**89**

**Figure 4.**

*following contact with water.*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

Within 48 hours after implantation in the human tissue, the Hyexpan cylinders absorb water to become thicker and shorter. The in vitro maximum diameter is 6.5 mm and the length is 17 mm (**Figure 4**). The volume of each individual implant

also becomes much softer in consistency. On the other hand, the SK prostheses in the dehydrated state are thin, solid cylinders, 29 mm long with a diameter of 3 mm, changing their size to a length of 23 mm and a diameter of 7 mm within 48 h of

The technique of implantation of the GK and SK is identical. The operation is performed under regional or general anaesthesia. Intravenous antibiotics are given at induction. The author's patients receive gentamicin 1.5 mg/kg and metronidazole 500 mg IV. The patient is placed in the lithotomy position. A strict sterile technique is used. The IAS and intersphincteric groove are identified by the placement of an anal retractor (e.g. Eisenhammer). The author's preference is a THD surgy Minilight proctoscope, a self-illuminating anal and rectal retractor that gives a very good view of the anorectum without causing trauma to the anal sphincter (**Figure 5**). A 2 mm incision is made in the perianal skin, 2 cm from the anal verge (**Figure 6**). Having attached the dispenser to the delivery system, the needle is inserted through the incision and tunnelled to the intersphincteric margin and introduced into the intersphincteric space. The needle is then positioned so that the tip would lie just beyond the dentate line. When the needle is identified in the correct position, by direct vision and palpation and/or by endoanal ultrasound, the prosthesis is

The steps may be repeated to insert up to 10 prostheses, equidistant from each other. The GK has been originally described with the insertion of between 4 and 6 prostheses, whereas the SK has been described with the use of 10 prostheses. The choice of inserting 4 as opposed to 6 or 10 prostheses is arbitrary. The use of 10 prostheses enables the formation of a circumferential or quasi-circumferential intersphincteric ring, akin to an artificial anal sphincter. The prostheses self-fix in the desired position, thereby preventing displacement and migration in the major-

The wounds are closed with a single absorbable suture (**Figure 8**). At the end of procedure, EAUS imaging will show the location of all prostheses. The procedure takes about 30 to 40 minutes to complete and is done as a day case. Oral metronidazole 400 mg tds is prescribed for 5 days postoperatively. Oral laxatives such as lactulose are prescribed to minimise the risk of constipation. The patients are advised to avoid any anal trauma as well as anal intercourse for at least 72 h after implant insertion. The patients are followed up after 6 weeks and 3 months thereafter. The material remains identifiable both by palpation and by endoanal ultrasonography in

*(a) Shape of Hyexpan gatekeeper cylinder at insertion. (b) Fully expanded Hyexpan gatekeeper cylinder* 

to 500 mm3

, a 750% increase. The implant

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

increases from approximately 70 mm3

released into the intersphincteric space (**Figure 7**).

the postoperative period (**Figures 9** and **10**).

contact with fluids.

ity of cases.

#### *Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

*Current Topics in Faecal Incontinence*

bulking agents:

• Perianal sepsis

• Rectal prolapse

• Pelvic radiotherapy

• Immunosuppression

made gun (**Figure 3**).

ulcerative colitis)

The main indications for the use of the GK and SK are passive faecal incontinence, secondary to IAS dysfunction or damage, where conservative measures or injection of other bulking agents such as PTQ or Permacol has failed. However, the

The following are contraindications to the use of the GK and SK. Similar contraindications have also been described by the product manufacturers of other anal

• Inflammatory bowel diseases with anorectal involvement (Crohn's disease,

use of GK and SK in patients with other causes of FI is being explored.

• Anal, rectal or colon cancer undergoing active treatment

• Pregnancy or planned pregnancy in the next 12 months.

Whereas the anal bulking agents that were developed in phases 1 and 2 are injected into or around the anal canal by means of a hypodermic syringe, the Hyexpan prostheses are implanted into the intersphincteric space using a custom-

The difference between GK and SK lies in the size of the prostheses. The dehydrated GK prostheses consist of thin solid cylinders, 22 mm long and 2 mm in diameter. The success of this material depends on its hydrophilic properties.

*The gatekeeper gun, made of the dispenser that houses one prosthesis and the delivery system. The Sphinkeeper* 

• Rectal bleeding of unknown or undiagnosed origin

• Uncontrolled blood coagulation disorders

*2.4.1 Surgical apparatus, procedure and technique*

*delivery system and dispenser are similar but slightly larger.*

**88**

**Figure 3.**

Within 48 hours after implantation in the human tissue, the Hyexpan cylinders absorb water to become thicker and shorter. The in vitro maximum diameter is 6.5 mm and the length is 17 mm (**Figure 4**). The volume of each individual implant increases from approximately 70 mm3 to 500 mm3 , a 750% increase. The implant also becomes much softer in consistency. On the other hand, the SK prostheses in the dehydrated state are thin, solid cylinders, 29 mm long with a diameter of 3 mm, changing their size to a length of 23 mm and a diameter of 7 mm within 48 h of contact with fluids.

The technique of implantation of the GK and SK is identical. The operation is performed under regional or general anaesthesia. Intravenous antibiotics are given at induction. The author's patients receive gentamicin 1.5 mg/kg and metronidazole 500 mg IV. The patient is placed in the lithotomy position. A strict sterile technique is used. The IAS and intersphincteric groove are identified by the placement of an anal retractor (e.g. Eisenhammer). The author's preference is a THD surgy Minilight proctoscope, a self-illuminating anal and rectal retractor that gives a very good view of the anorectum without causing trauma to the anal sphincter (**Figure 5**). A 2 mm incision is made in the perianal skin, 2 cm from the anal verge (**Figure 6**).

Having attached the dispenser to the delivery system, the needle is inserted through the incision and tunnelled to the intersphincteric margin and introduced into the intersphincteric space. The needle is then positioned so that the tip would lie just beyond the dentate line. When the needle is identified in the correct position, by direct vision and palpation and/or by endoanal ultrasound, the prosthesis is released into the intersphincteric space (**Figure 7**).

The steps may be repeated to insert up to 10 prostheses, equidistant from each other. The GK has been originally described with the insertion of between 4 and 6 prostheses, whereas the SK has been described with the use of 10 prostheses. The choice of inserting 4 as opposed to 6 or 10 prostheses is arbitrary. The use of 10 prostheses enables the formation of a circumferential or quasi-circumferential intersphincteric ring, akin to an artificial anal sphincter. The prostheses self-fix in the desired position, thereby preventing displacement and migration in the majority of cases.

The wounds are closed with a single absorbable suture (**Figure 8**). At the end of procedure, EAUS imaging will show the location of all prostheses. The procedure takes about 30 to 40 minutes to complete and is done as a day case. Oral metronidazole 400 mg tds is prescribed for 5 days postoperatively. Oral laxatives such as lactulose are prescribed to minimise the risk of constipation. The patients are advised to avoid any anal trauma as well as anal intercourse for at least 72 h after implant insertion. The patients are followed up after 6 weeks and 3 months thereafter. The material remains identifiable both by palpation and by endoanal ultrasonography in the postoperative period (**Figures 9** and **10**).

#### **Figure 4.**

*(a) Shape of Hyexpan gatekeeper cylinder at insertion. (b) Fully expanded Hyexpan gatekeeper cylinder following contact with water.*

#### **Figure 5.**

*Palpating the IAS and the intersphincteric groove at the 6 o'clock position with a THD surgy mini-light proctoscope in position.*

**91**

**Figure 8.**

**Figure 7.**

*correct placement.*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

*The gatekeeper needle at the 9 o'clock position, with the endoanal ultrasound probe in place to determine* 

*Up to 10 equidistant circumferential perianal wounds, each closed with an absorbable suture (Monocryl 3/0).*

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

**Figure 6.** *Making an incision, 2 cm away from the anal verge, at the 6 o'clock position.*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

#### **Figure 7.**

*Current Topics in Faecal Incontinence*

**90**

**Figure 6.**

**Figure 5.**

*proctoscope in position.*

*Making an incision, 2 cm away from the anal verge, at the 6 o'clock position.*

*Palpating the IAS and the intersphincteric groove at the 6 o'clock position with a THD surgy mini-light* 

*The gatekeeper needle at the 9 o'clock position, with the endoanal ultrasound probe in place to determine correct placement.*

#### **Figure 9.**

*Endoanal ultrasound scan (Aloka) at 6 weeks following the implantation of six gatekeeper prostheses in a 72-year-old male with idiopathic passive faecal incontinence.*

#### *2.4.2 Results*

The first reported experience with the Gatekeeper was by Ratto et al. in 2011 [19]. This was a study with 14 patients. Eight had idiopathic FI, four had an IAS defect, and two had combined IAS and EAS defects. The median follow-up was of 12 months (ranging from 5 to 48 months). The authors reported a clinically significant improvement in continence in 13 patients, a sustained significant improvement in the Wexner and Vaizey scores and in the SF36 and FIQOL scores. No complications have been reported.

The second study was a comparative retrospective study by Parello et al. in 2012 [20]. Seven patients who had the Gatekeeper implanted were compared to six patients who underwent sacral nerve stimulation. The median follow-up was of 18 months in the Gatekeeper group and 20 months in the SNS group. The authors reported a sustained improvement in the Wexner continence scores with both modalities of treatment.

Fabiani et al. [21] used Gatekeeper for a group of patients affected by minor faecal incontinence. Four out of seven patients complained of passive incontinence prior to the procedure. After an average follow-up of 6 months, 6 patients reported a Wexner incontinence score under the value of 4, meaning that they rarely experienced symptoms (0 = perfect incontinence and 20 = complete incontinence). Only one patient who suffered mixed incontinence failed to respond.

Biondo et al. [22] concluded that Gatekeeper is a safe and effective procedure in more than 50% of patients for at least a year after implantation. They found that no patients had postoperative or long-term complications. Forty-eight per cent of patients were classed as responders, and significant differences were found between baseline mean Vaizey scores at 6 months, 12 months and last follow-up. At long-term follow-up (2.7 years), those patients that responded were found to have maintained an improvement more than 50% of their baseline Vaizey score.

**93**

continence.

**Figure 10.**

is managed by paracetamol.

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

In a multicentre study involving 54 patients and a clinical follow-up for a year, Ratto et al. [23] noted that after Gatekeeper implantation, incontinence to gas, liquid and solid stool improved significantly, soiling was reduced and the ability to defer defaecation was enhanced. All faecal incontinence severity scores were significantly reduced, and patients' quality of life improved. At 12 months, 30 patients (56 per cent) showed at least 75 per cent improvement in all faecal incontinence parameters, and 7 (13 per cent) became fully continent. Dislodgement of a few prostheses was reported, but this made no difference to postoperative

*Endoanal ultrasound scan (B&K) at 6 weeks following the implantation of 10 Sphinkeeper prostheses in a* 

*68-year-old female with passive faecal incontinence and previous episiotomy.*

The author has carried out more than 40 GK procedures in a single centre since 2012. The main indications were idiopathic FI and passive incontinence following surgery (anal stretch for anal fissure and haemorrhoidectomy). All patients had failed conservative management. There was a significant sustained improvement in the median Vaizey scores. The median (range) Vaizey scores improved from 16 (12–17) preoperatively to 5 (3–9), 4 (3–7), 4 (3–5), 4 (3–5), 5 (3–6) and 5 (3–6) at 6 weeks and at 3, 6, 12, 24 and 36 months, respectively (p < 0.01, Wilcoxon test). There was also an improvement in the Rockwood quality of life scores. The author reports no complications apart from minor pain that

Publications on the Sphinkeeper are limited. Ratto et al. [24] treated 10 patients with SK and followed them up for 3 months. The study demonstrated that the SK, with its larger prostheses than that of GK, is safe and effective. The Pelvic Floor Society of the Association of Coloproctology of Great Britain and Ireland is currently collecting prospective data on the SK from multiple centres in the UK.

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

#### **Figure 10.**

*Current Topics in Faecal Incontinence*

**92**

*2.4.2 Results*

**Figure 9.**

tions have been reported.

*72-year-old male with idiopathic passive faecal incontinence.*

modalities of treatment.

The first reported experience with the Gatekeeper was by Ratto et al. in 2011 [19]. This was a study with 14 patients. Eight had idiopathic FI, four had an IAS defect, and two had combined IAS and EAS defects. The median follow-up was of 12 months (ranging from 5 to 48 months). The authors reported a clinically significant improvement in continence in 13 patients, a sustained significant improvement in the Wexner and Vaizey scores and in the SF36 and FIQOL scores. No complica-

*Endoanal ultrasound scan (Aloka) at 6 weeks following the implantation of six gatekeeper prostheses in a* 

The second study was a comparative retrospective study by Parello et al. in 2012 [20]. Seven patients who had the Gatekeeper implanted were compared to six patients who underwent sacral nerve stimulation. The median follow-up was of 18 months in the Gatekeeper group and 20 months in the SNS group. The authors reported a sustained improvement in the Wexner continence scores with both

Fabiani et al. [21] used Gatekeeper for a group of patients affected by minor faecal incontinence. Four out of seven patients complained of passive incontinence prior to the procedure. After an average follow-up of 6 months, 6 patients reported a Wexner incontinence score under the value of 4, meaning that they rarely experienced symptoms (0 = perfect incontinence and 20 = complete incontinence). Only

Biondo et al. [22] concluded that Gatekeeper is a safe and effective procedure in more than 50% of patients for at least a year after implantation. They found that no patients had postoperative or long-term complications. Forty-eight per cent of patients were classed as responders, and significant differences were found between baseline mean Vaizey scores at 6 months, 12 months and last follow-up. At long-term follow-up (2.7 years), those patients that responded were found to have

maintained an improvement more than 50% of their baseline Vaizey score.

one patient who suffered mixed incontinence failed to respond.

*Endoanal ultrasound scan (B&K) at 6 weeks following the implantation of 10 Sphinkeeper prostheses in a 68-year-old female with passive faecal incontinence and previous episiotomy.*

In a multicentre study involving 54 patients and a clinical follow-up for a year, Ratto et al. [23] noted that after Gatekeeper implantation, incontinence to gas, liquid and solid stool improved significantly, soiling was reduced and the ability to defer defaecation was enhanced. All faecal incontinence severity scores were significantly reduced, and patients' quality of life improved. At 12 months, 30 patients (56 per cent) showed at least 75 per cent improvement in all faecal incontinence parameters, and 7 (13 per cent) became fully continent. Dislodgement of a few prostheses was reported, but this made no difference to postoperative continence.

The author has carried out more than 40 GK procedures in a single centre since 2012. The main indications were idiopathic FI and passive incontinence following surgery (anal stretch for anal fissure and haemorrhoidectomy). All patients had failed conservative management. There was a significant sustained improvement in the median Vaizey scores. The median (range) Vaizey scores improved from 16 (12–17) preoperatively to 5 (3–9), 4 (3–7), 4 (3–5), 4 (3–5), 5 (3–6) and 5 (3–6) at 6 weeks and at 3, 6, 12, 24 and 36 months, respectively (p < 0.01, Wilcoxon test). There was also an improvement in the Rockwood quality of life scores. The author reports no complications apart from minor pain that is managed by paracetamol.

Publications on the Sphinkeeper are limited. Ratto et al. [24] treated 10 patients with SK and followed them up for 3 months. The study demonstrated that the SK, with its larger prostheses than that of GK, is safe and effective. The Pelvic Floor Society of the Association of Coloproctology of Great Britain and Ireland is currently collecting prospective data on the SK from multiple centres in the UK.

#### **3. Discussion**

The development of anal injectable and implantable technology over the past 20 years has taken great strides forwards. Starting with the pioneering efforts of Shafik with autologous fat, more materials have been tried and used, the most popular being collagen (Permacol) and silicone (PTQ or Bioplastique). These agents were associated with variable and inconsistent results. Injections were frequently repeated to maintain continence in the long term. The latest generation of anal bulking agents is the implantable Hyexpan (Gatekeeper and Sphinkeeper). This material fits the criteria for the 'ideal' bulking agent. It overcomes most limitations of other bulking agents, and its use has shown very promising results.

The choice to implant the GK and SK prostheses into the intersphincteric space of the anal canal plays a key role. This location potentially avoids extrusion or migration of prostheses (different to what could happen if implanted into the submucosa). Moreover, thanks to the rapid increase of their volume, the prostheses self-fix and are unlikely to move after deployment.

The mechanism of action of anal bulking agents is a subject of debate. Most of the resting anal pressure is the function of the IAS, with some contribution from the EAS and anal cushions. Studies of faecal incontinence in patients who have undergone a traditional Milligan-Morgan haemorrhoidectomy lend support to the concept that anal cushions play an important part in the maintenance of the normal mechanism of continence. It is thought that the mechanism of action of a bulking agent injected into the submucosal space is an increase in the size of the natural anal cushions. On the other hand, a bulking agent injected or implanted into the intersphincteric space would bulk up the size of the anal sphincter. The end result would be an improvement in the seal of the lumen of the anal canal at rest and potentially an increase in resting anal pressure and in the length of the anal high pressure zone. When the injection is placed adjacent to an identifiable IAS defect, a better degree of anal canal sealing may be obtained through improvement in the configuration and symmetry of the anal canal [7]. Ratto argues that GK and SK, being embedded within the intersphincteric space, thereby pushing the EAS outwards and the IAS inwards, 'may improve sphincter contractility by increasing sarcomere length as well as increase the length of the anal canal and provide a powerful "bulking effect"' [24].

It is acknowledged that more research is required in this field. Most studies are case series with very few randomised trials. The Gatekeeper and Sphinkeeper, the latest generation of anal bulking agents, show promising results. Whether these results are maintained in the longer term or not awaits to be seen. The key factor however remains that correct patient selection is extremely important to achieve good results.

Larger series with longer follow-up and randomised controlled trials are therefore necessary. Further development on existing and emerging technology is also warranted.

**95**

**Author details**

John Camilleri-Brennan

Medical School, Scotland, UK

provided the original work is properly cited.

Department of Surgery, Forth Valley Royal Hospital and the University of Glasgow

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: johncbrennan@doctors.org.uk

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence*

*DOI: http://dx.doi.org/10.5772/intechopen.91952*

*Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

*Current Topics in Faecal Incontinence*

The development of anal injectable and implantable technology over the past 20 years has taken great strides forwards. Starting with the pioneering efforts of Shafik with autologous fat, more materials have been tried and used, the most popular being collagen (Permacol) and silicone (PTQ or Bioplastique). These agents were associated with variable and inconsistent results. Injections were frequently repeated to maintain continence in the long term. The latest generation of anal bulking agents is the implantable Hyexpan (Gatekeeper and Sphinkeeper). This material fits the criteria for the 'ideal' bulking agent. It overcomes most limitations

of other bulking agents, and its use has shown very promising results.

self-fix and are unlikely to move after deployment.

The choice to implant the GK and SK prostheses into the intersphincteric space of the anal canal plays a key role. This location potentially avoids extrusion or migration of prostheses (different to what could happen if implanted into the submucosa). Moreover, thanks to the rapid increase of their volume, the prostheses

The mechanism of action of anal bulking agents is a subject of debate. Most of the resting anal pressure is the function of the IAS, with some contribution from the EAS and anal cushions. Studies of faecal incontinence in patients who have undergone a traditional Milligan-Morgan haemorrhoidectomy lend support to the concept that anal cushions play an important part in the maintenance of the normal mechanism of continence. It is thought that the mechanism of action of a bulking agent injected into the submucosal space is an increase in the size of the natural anal cushions. On the other hand, a bulking agent injected or implanted into the intersphincteric space would bulk up the size of the anal sphincter. The end result would be an improvement in the seal of the lumen of the anal canal at rest and potentially an increase in resting anal pressure and in the length of the anal high pressure zone. When the injection is placed adjacent to an identifiable IAS defect, a better degree of anal canal sealing may be obtained through improvement in the configuration and symmetry of the anal canal [7]. Ratto argues that GK and SK, being embedded within the intersphincteric space, thereby pushing the EAS outwards and the IAS inwards, 'may improve sphincter contractility by increasing sarcomere length as well as increase the length of the anal canal and provide a powerful "bulking

It is acknowledged that more research is required in this field. Most studies are case series with very few randomised trials. The Gatekeeper and Sphinkeeper, the latest generation of anal bulking agents, show promising results. Whether these results are maintained in the longer term or not awaits to be seen. The key factor however remains that correct patient selection is extremely important to achieve

Larger series with longer follow-up and randomised controlled trials are therefore necessary. Further development on existing and emerging technology is also

**3. Discussion**

**94**

effect"' [24].

good results.

warranted.

#### **Author details**

John Camilleri-Brennan

Department of Surgery, Forth Valley Royal Hospital and the University of Glasgow Medical School, Scotland, UK

\*Address all correspondence to: johncbrennan@doctors.org.uk

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[3] Rockwood TH, Church JM, Fleshman JW, et al. Fecal incontinence quality of life scale: Quality of life instrument for patients with fecal incontinence. Diseases of the Colon and Rectum. 2000;**43**(1):9-17

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[22] Biondo S, Trenti L, Noguerales F, Nomdedeu J, et al. Outcomes of gatekeeper prosthesis implantation for the treatment of fecal incontinence: A multicenter observational study. Techniques in Coloproctology.

[23] Ratto C, Buntzen S, Aigner F, Altomare DF, et al. Multicentre observational study of the gatekeeper for faecal incontinence. British Journal

of Surgery. 2016;**103**:290-299

[24] Ratto C, Donisi L, Litta F, Campennı P, Parello A. Implantation of SphinKeeper: A new artificial anal sphincter. Techniques in Coloproctology. 2016;**20**:59-66

Donisi L, Litta F, Zaccone G, Ratto C. Can gatekeeper, a new bulking agent be equivalent to SNS in patients with fecal incontinence in the presence of only internal sphincter defects. Techniques in *Anal Injectable and Implantable Bulking Agents for Faecal Incontinence DOI: http://dx.doi.org/10.5772/intechopen.91952*

reaction after silicone injection for fecal incontinence in humans: Two case reports. Techniques in Coloproctology. 2012;**16**:395-397

[19] Ratto C, Parello A, Donisi L, Litta F, De Simone V, Spazzafumo L, et al. Novel bulking agent for faecal incontinence. British Journal of Surgery. 2011;**98**(11):1644-1652

[20] Parello A, De Simone V, Donisi L, Litta F, Zaccone G, Ratto C. Can gatekeeper, a new bulking agent be equivalent to SNS in patients with fecal incontinence in the presence of only internal sphincter defects. Techniques in Coloproctology. 2012;**16**:77

[21] Fabiani B, Menconi CDE, Giani I, Naldini G. Anal gatekeeper a new bulking agent for faecal incontinence. Colorectal Disease;**16**(Suppl 3):37-105

[22] Biondo S, Trenti L, Noguerales F, Nomdedeu J, et al. Outcomes of gatekeeper prosthesis implantation for the treatment of fecal incontinence: A multicenter observational study. Techniques in Coloproctology. 2017;**21**:963-970

[23] Ratto C, Buntzen S, Aigner F, Altomare DF, et al. Multicentre observational study of the gatekeeper for faecal incontinence. British Journal of Surgery. 2016;**103**:290-299

[24] Ratto C, Donisi L, Litta F, Campennı P, Parello A. Implantation of SphinKeeper: A new artificial anal sphincter. Techniques in Coloproctology. 2016;**20**:59-66

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[1] Vaizey CJ, Carapeti E, Cahill JA, Kamm MA. Prospective comparison of faecal incontinence grading systems.

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[11] Maeda Y, Laurberg S, Norton C. Perianal injectable bulking agents as treatment for faecal incontinence in adults. Cochrane Database of Systematic

[12] Watson NFS, Koshy A, Sagar PM.

Reviews. 2010;**5**:CD007959

Anal bulking agents for faecal incontinence. Colorectal Disease.

[13] Tjandra JJ, Lim JF, Hiscock R, Rajendra P. Injectable silicone biomaterial for fecal incontinence caused by internal anal sphincter dysfunction is effective. Diseases of the Colon and Rectum. 2004;**47**:2138-2146

[14] Tjandra JJ, Chan MK, Yeh HC. Injectable silicone biomaterial (PTQ ) is more effective than carbon-coated beads (Durasphere) in treating passive faecal incontinence-a randomized trial. Colorectal Disease. 2009;**11**(4):382-389

[15] Siproudhis L, Morcet J,

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Laine F. Elastomer implants in faecal incontinence: A blind, randomized placebo-controlled study. Alimentary Pharmacology & Therapeutics.

[16] Graf W, Mellgren A, Matzel KE, Hull T, Johansson C, Bernstein M, et al. Efficacy of dextranomer in stabilised hyaluronic acid for treatment of faecal incontinence: A randomised, sham-controlled trial. Lancet. 2011;**377**(9770):997-1003

[17] Maeda Y, Vaizey CJ, Kamm MA. Pilot study of two new injectable bulking agents for the treatment of faecal incontinence. Colorectal Disease.

[18] van Wunnik B, Driessen A, Baeten C. Local giant cell foreign body

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[2] Jorge JMN, Wexner SD. Etiology and management of fecal incontinence. Diseases of the Colon and Rectum.

[3] Rockwood TH, Church JM,

Rectum. 2000;**43**(1):9-17

1992;**30**:473-483

Fleshman JW, et al. Fecal incontinence quality of life scale: Quality of life instrument for patients with fecal incontinence. Diseases of the Colon and

[4] Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Medical Care.

[5] National Institute for Clinical

Guideline 49 NICE; 2007

Surgery. 2005;**92**:521-527

Surgery. 1993;**78**:159-191

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[6] Lightner DJ. Review of the available urethral bulking agents. Current Opinion in Urology. 2002;**12**:333-338

[7] Vaizey CJ, Kamm MA. Injectable bulking agents for treating faecal incontinence. The British Journal of

[8] Shafik A. Polytetrafluoroethylene injection for the treatment of partial fecal incontinence. International

[9] Shafik A. Perianal injection of autologous fat for treatment of sphincteric incontinence. Diseases of the Colon and Rectum. 1995;**38**:583-587

[10] Bernardi C, Favetta U, Pescatori M. Autologous fat injection for treatment of fecal incontinence: Manometric and echographicassessment.

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1993;**36**:77-97

**Chapter 7**

**Abstract**

BioSphincter a Regenerative

*Prabhash Dadhich and Khalil N. Bitar*

Medicine Approach to Treat FI

A healthy sphincter physiology is a complex interplay between neural and muscle population, responsible for relaxation and contraction, which allow feces to pass and reestablishment of closure. The loss of integrity of neuromuscular functionality or cellular component results in fecal incontinence (FI). The current available treatments have been disappointing in long-term relief. This chapter represents a regenerative medicine approach to this debilitating disease, wherein a new internal anal sphincter (IAS) BioSphincter™ is bioengineered from the patient's own cells and implanted. It results in long-term restoration of the cellular integrity and reinstatement of the physiological function of the IAS. Following implantation in rodents, the engineered sphincters became vascularized and maintained their phenotype and functionality. The developed IAS BioSphincter™ were validated to treat the FI in large animals and successfully restored anorectal functionality. According to NIH/NIDDK, one out of seven people report to health care providers complaining of fecal incontinence. This chapter elucidates the long road in developing on implantable bioengineered IAS "BioSphincter™" that would benefit and improve the quality of life of a large socially distressed segment of the population.

**Keywords:** fecal incontinence, regenerative medicine, BioSphincters, neural

A healthy anorectal functionality is a coordinated interplay between the enteric nervous system, smooth muscle of internal anal sphincter (IAS), striated external anal sphincter and puborectalis muscles [1]. Anomalies in any of this individual or group of tissues may lead to anorectal irregularities and diseases [2]. Fecal incontinence (FI) is devastating from a hygiene perspective due to involuntary soiling of liquid and solid stool and results in the distressing psychosocial impact on the patient [3]. Injury to the perineum may also result in the complete or partial destruction of the anal sphincter and distal rectum potentially resulting in persistent incontinence [4]. The resulting psychological stress, social stigma, decreased selfesteem and productivity can be overwhelming. In the USA, men and women suffer from FI equally with a range of 2–6% in people aged 20–30 years. The prevalence

Clinical characteristics of FI have been correlated with underlying sphincter pathology [1]. In the classical FI, the pelvic floor muscles are dysfunctional (due to muscle or nerve damage) and result in the frequent urge of incontinence. The urge of incontinence is mainly due to external anal sphincter defects and lower anorectal

progenitor cells, anorectal physiology

increases to over 15% in people older than 70 years [5].

**1. Introduction**

**99**

**Chapter 7**

## BioSphincter a Regenerative Medicine Approach to Treat FI

*Prabhash Dadhich and Khalil N. Bitar*

#### **Abstract**

A healthy sphincter physiology is a complex interplay between neural and muscle population, responsible for relaxation and contraction, which allow feces to pass and reestablishment of closure. The loss of integrity of neuromuscular functionality or cellular component results in fecal incontinence (FI). The current available treatments have been disappointing in long-term relief. This chapter represents a regenerative medicine approach to this debilitating disease, wherein a new internal anal sphincter (IAS) BioSphincter™ is bioengineered from the patient's own cells and implanted. It results in long-term restoration of the cellular integrity and reinstatement of the physiological function of the IAS. Following implantation in rodents, the engineered sphincters became vascularized and maintained their phenotype and functionality. The developed IAS BioSphincter™ were validated to treat the FI in large animals and successfully restored anorectal functionality. According to NIH/NIDDK, one out of seven people report to health care providers complaining of fecal incontinence. This chapter elucidates the long road in developing on implantable bioengineered IAS "BioSphincter™" that would benefit and improve the quality of life of a large socially distressed segment of the population.

**Keywords:** fecal incontinence, regenerative medicine, BioSphincters, neural progenitor cells, anorectal physiology

#### **1. Introduction**

A healthy anorectal functionality is a coordinated interplay between the enteric nervous system, smooth muscle of internal anal sphincter (IAS), striated external anal sphincter and puborectalis muscles [1]. Anomalies in any of this individual or group of tissues may lead to anorectal irregularities and diseases [2]. Fecal incontinence (FI) is devastating from a hygiene perspective due to involuntary soiling of liquid and solid stool and results in the distressing psychosocial impact on the patient [3]. Injury to the perineum may also result in the complete or partial destruction of the anal sphincter and distal rectum potentially resulting in persistent incontinence [4]. The resulting psychological stress, social stigma, decreased selfesteem and productivity can be overwhelming. In the USA, men and women suffer from FI equally with a range of 2–6% in people aged 20–30 years. The prevalence increases to over 15% in people older than 70 years [5].

Clinical characteristics of FI have been correlated with underlying sphincter pathology [1]. In the classical FI, the pelvic floor muscles are dysfunctional (due to muscle or nerve damage) and result in the frequent urge of incontinence. The urge of incontinence is mainly due to external anal sphincter defects and lower anorectal squeeze pressures. Patients with the urge of incontinence have FI episodes with awareness of the event but cannot prevent it because of the inability to increase anorectal pressures [1, 6, 7].

However, a 3-month follow up study of injection of dextranomer microspheres

Cell delivery is advanced translation method for long-term efficacy in FI. Stem cell constructs were developed, and were able to generate smooth muscle tone but lacked innervation [25]. Autologous transplantation of muscle progenitor cells into the sphincters exhibited potentials for re-stabilization of myogenic functionality in the anal sphincters [26]. Delivery of autologous human adipose-derived stem cells in poorly functioning sphincter muscle as replacement of fibrous tissues acted as a mechanical support for physiological functions [27]. Injection of autologous myoblasts into the external anal sphincter defect also resulted as a safe and promising approach to improve symptoms of FI induced owing to obstetric anal sphincter trauma [28]. Sphincters are complex organs for cell delivery. There are several challenges to overcome in direct cell delivery, such as specific types and dosages of cells, circular distribution and orientation of cells around the anal canal after injection, functional integration with host cells and

Current cell delivery technologies focus either on the reinstatement of the striated muscle of the external anal sphincter or mechanical support to the sphincter, with little attention on the reinstatement of IAS function [29–32]. The terminal gut function requires coordinated contraction and relaxation of the smooth muscle of rectum mediated through the enteric nervous system of IAS [2, 6]. To remedy an injured anus, it is imperative to reinstate both smooth muscle and intrinsic neural components of IAS. We describe the evolution of a regenerative medicine approach proposed to provide critical components to reinstate function in the anorectum and remedy passive fecal incontinence caused by injury to the IAS. According to this hypothesis, implantation of engineered autologous BioSphincters reinstate IAS function and restore fecal continence. Autologous smooth muscle and neural progenitor cells from gut biopsies were used to bioengineer intrinsically innervated IAS [33, 34]. Autologous functional intrinsically innervated IAS construct was successfully implanted into healthy animal models. Following implantation in rodents, the engineered sphincters became vascularized and maintained their phenotype and functionality [35–38]. A large animal model of passive fecal incontinence was developed and demonstrated sustained restoration of fecal continence, and restoration of basal tone and restoration of RAIR after implantation of engineered autologous, intrinsically innervated internal

resulted in a 50% reduction in FI frequency in 52% patients [24].

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

long-term effects such as biodistribution, tumorigenicity.

anal sphincter (IAS) BioSphincters [10, 39] (**Figure 1**).

*Regenerative medicine approach to treat fecal incontinence using autologous bioengineered BioSphincter.*

**Figure 1.**

**101**

The passive FI caused an isolated or combined loss of smooth muscle function (IAS), skeletal muscle function (EAS), anorectal sensory mechanisms or neural control [8, 9]. It leads to loss of the sense that rectum is full and results in unknowingly leakage of stools, mucus, and flatus. Passive incontinence occurs without the patient's awareness of the event until after incontinence has occurred [6]. Patients with passive incontinence are more likely to have internal anal sphincter defects and lower anorectal resting pressures. The anal resting tone is produced by the internal anal sphincter (IAS) and the external anal sphincter. The IAS contributes 60–70% of the anal tone [10]. In addition, patients with passive incontinence have been shown to have more frequent and exaggerated IAS relaxation compared to continent controls [11]. Patients with FI have been shown to have variable loss of the Recto Anal Inhibitory Reflex (RAIR) [12].

Currently, there is no satisfactory long-term treatment for FI. Epidemiological studies indicated that most patients suffering from FI do not consult to clinicians and depend on self-management or rely on the use of adult diapers. The classical treatment of FI becomes more involved in accordance with the extent and severity of the incidences of incontinence.

Conservative management of FI is usually initiated with educating the patients with behavioral techniques. These techniques such as scheduling toileting and preventive strategies [13]. The next step is the incorporation of dietary changes using fiber supplements or laxative to normalize stool consistency [14]. Along with dietary modulations, antidiarrheal drugs, alpha (1 and 2) receptor agonists could also be used to control the frequency of FI episodes [15]. Pelvic floor muscle exercise and biofeedback are other conservative methods to manage initial stages of FI. Biofeedback methods are behavioral management that incorporates electronic and mechanical devices to emphasize bowel and muscle retraining. Pelvic floor muscle exercises with biofeedback improve sense and strength of pelvic floor muscles for contraction during rectal distention and uncontrollable urge of FI [16, 17]. According to an observational study, these conservative methods resulted in 50% reduction in the frequency of FI and 21% adequate relief in FI [13]. The effectiveness and success of these measures may help in the management of mild cases of FI.

If the patient does not improve with the mentioned conservative methods, the patient is offered advanced therapies. Advanced therapies are more invasive and involve different levels of surgical interventions such as electrical stimulation, sphincteroplasty, injection of bulking agents, and implantable devices. Sacral nerve and tibial nerve stimulation found to be more effective than electrical stimulation of muscles [18–20]. In a randomized controlled trial on patients with structurally intact and innervated sphincters, the implantation of a battery-operated stimulator was found to be effective from 36 to 50% [18, 19]. The frequency of episodes of FI was reduced during stimulation, but unaffected without stimulation or similar to sham [13]. The implantable devices such as artificial bowel sphincter [21], magnetic beads [22] and synthetic polymer rings are implanted around the anal canal to augment the pressure. There is a lack of randomized controlled trial towards longterm safety and efficacy of these procedures. The sphincteroplasties (suturing of the separated sphincter) and graciloplasty (wrapping of gracilis muscle around the anal canal) are another class of surgical procedures to treat FI. These procedures have shown varying rates of success and high chances of obstructed defecation. Inert materials (silicone elastomers, ceramic beads) or biopolymers (polycaprolactam beads) as bulking agents injected around the anal canal to increase resting pressure [23]. There was no specific success reported regarding long-term efficacy.

#### *BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*

squeeze pressures. Patients with the urge of incontinence have FI episodes with awareness of the event but cannot prevent it because of the inability to increase

The passive FI caused an isolated or combined loss of smooth muscle function (IAS), skeletal muscle function (EAS), anorectal sensory mechanisms or neural control [8, 9]. It leads to loss of the sense that rectum is full and results in unknowingly leakage of stools, mucus, and flatus. Passive incontinence occurs without the patient's awareness of the event until after incontinence has occurred [6]. Patients with passive incontinence are more likely to have internal anal sphincter defects and lower anorectal resting pressures. The anal resting tone is produced by the internal anal sphincter (IAS) and the external anal sphincter. The IAS contributes 60–70% of the anal tone [10]. In addition, patients with passive incontinence have been shown to have more frequent and exaggerated IAS relaxation compared to continent controls [11]. Patients with FI have been shown to have variable loss of

Currently, there is no satisfactory long-term treatment for FI. Epidemiological studies indicated that most patients suffering from FI do not consult to clinicians and depend on self-management or rely on the use of adult diapers. The classical treatment of FI becomes more involved in accordance with the extent and severity

Conservative management of FI is usually initiated with educating the patients with behavioral techniques. These techniques such as scheduling toileting and preventive strategies [13]. The next step is the incorporation of dietary changes using fiber supplements or laxative to normalize stool consistency [14]. Along with dietary modulations, antidiarrheal drugs, alpha (1 and 2) receptor agonists could also be used to control the frequency of FI episodes [15]. Pelvic floor muscle exercise and biofeedback are other conservative methods to manage initial stages of FI. Biofeed-

back methods are behavioral management that incorporates electronic and

contraction during rectal distention and uncontrollable urge of FI [16, 17]. According to an observational study, these conservative methods resulted in 50% reduction in the frequency of FI and 21% adequate relief in FI [13]. The effectiveness and success of these measures may help in the management of mild cases of FI. If the patient does not improve with the mentioned conservative methods, the patient is offered advanced therapies. Advanced therapies are more invasive and involve different levels of surgical interventions such as electrical stimulation, sphincteroplasty, injection of bulking agents, and implantable devices. Sacral nerve and tibial nerve stimulation found to be more effective than electrical stimulation of muscles [18–20]. In a randomized controlled trial on patients with structurally intact and innervated sphincters, the implantation of a battery-operated stimulator was found to be effective from 36 to 50% [18, 19]. The frequency of episodes of FI was reduced during stimulation, but unaffected without stimulation or similar to sham [13]. The implantable devices such as artificial bowel sphincter [21], magnetic beads [22] and synthetic polymer rings are implanted around the anal canal to augment the pressure. There is a lack of randomized controlled trial towards longterm safety and efficacy of these procedures. The sphincteroplasties (suturing of the separated sphincter) and graciloplasty (wrapping of gracilis muscle around the anal canal) are another class of surgical procedures to treat FI. These procedures have shown varying rates of success and high chances of obstructed defecation. Inert materials (silicone elastomers, ceramic beads) or biopolymers (polycaprolactam beads) as bulking agents injected around the anal canal to increase resting pressure

[23]. There was no specific success reported regarding long-term efficacy.

mechanical devices to emphasize bowel and muscle retraining. Pelvic floor muscle exercises with biofeedback improve sense and strength of pelvic floor muscles for

anorectal pressures [1, 6, 7].

*Current Topics in Faecal Incontinence*

the Recto Anal Inhibitory Reflex (RAIR) [12].

of the incidences of incontinence.

**100**

However, a 3-month follow up study of injection of dextranomer microspheres resulted in a 50% reduction in FI frequency in 52% patients [24].

Cell delivery is advanced translation method for long-term efficacy in FI. Stem cell constructs were developed, and were able to generate smooth muscle tone but lacked innervation [25]. Autologous transplantation of muscle progenitor cells into the sphincters exhibited potentials for re-stabilization of myogenic functionality in the anal sphincters [26]. Delivery of autologous human adipose-derived stem cells in poorly functioning sphincter muscle as replacement of fibrous tissues acted as a mechanical support for physiological functions [27]. Injection of autologous myoblasts into the external anal sphincter defect also resulted as a safe and promising approach to improve symptoms of FI induced owing to obstetric anal sphincter trauma [28]. Sphincters are complex organs for cell delivery. There are several challenges to overcome in direct cell delivery, such as specific types and dosages of cells, circular distribution and orientation of cells around the anal canal after injection, functional integration with host cells and long-term effects such as biodistribution, tumorigenicity.

Current cell delivery technologies focus either on the reinstatement of the striated muscle of the external anal sphincter or mechanical support to the sphincter, with little attention on the reinstatement of IAS function [29–32]. The terminal gut function requires coordinated contraction and relaxation of the smooth muscle of rectum mediated through the enteric nervous system of IAS [2, 6]. To remedy an injured anus, it is imperative to reinstate both smooth muscle and intrinsic neural components of IAS. We describe the evolution of a regenerative medicine approach proposed to provide critical components to reinstate function in the anorectum and remedy passive fecal incontinence caused by injury to the IAS. According to this hypothesis, implantation of engineered autologous BioSphincters reinstate IAS function and restore fecal continence. Autologous smooth muscle and neural progenitor cells from gut biopsies were used to bioengineer intrinsically innervated IAS [33, 34]. Autologous functional intrinsically innervated IAS construct was successfully implanted into healthy animal models. Following implantation in rodents, the engineered sphincters became vascularized and maintained their phenotype and functionality [35–38]. A large animal model of passive fecal incontinence was developed and demonstrated sustained restoration of fecal continence, and restoration of basal tone and restoration of RAIR after implantation of engineered autologous, intrinsically innervated internal anal sphincter (IAS) BioSphincters [10, 39] (**Figure 1**).

**Figure 1.**

*Regenerative medicine approach to treat fecal incontinence using autologous bioengineered BioSphincter.*

This chapter summarizes the regenerative medicine approach of bioengineering of BioSphincters, including developmental stages of the technology, challenges, process optimization, characterization, detail pre-clinical evaluation of the BioSphincter towards the treatment of FI.

In this endeavor, smooth muscle cells were isolated from the IAS of donor C57BL/6 mice. Smooth muscle cell constructs were engineered on Sylgard coated plates using fibrin gel, as described previously [40]. The engineered constructs were successfully implanted into the subcutaneous region of same strain mice and treated with either fibroblastic growth factor-2 or saline as controls using a microosmotic pump. Mice were euthanized after 4 weeks, and the implant was harvested. The implant was intact, healthy in color without any degradation, and interestingly displayed muscle attachment to the back of the mouse, with neovascularization. Constructs exhibited no external sign of inflammation, fibrosis, or infection, because of the use of syngeneic tissue. The supplement of FGF-2 also helped in tissue viability, cellular integrity, and vascularization. The harvested tissues

The post-implant harvested constructs were analyzed for force generation. The harvested implants generated and maintained the spontaneous basal tone in the absence of any external stimuli. The developed tone confirmed the integrity of ionic membrane characteristics, membrane receptors and their intracellular signaling mechanisms for contraction and relaxation. On treatment of a relaxing stimulant such as a vasoactive intestinal peptide (VIP), the force and magnitude of relaxation were consistent before and after implantation. The rapid, and dose-dependent sustained (over 30 min without signs of muscle fatigue) contractions on the treatment of acetylcholine and phorbol dibutyrate was elicited as well. The physiological studies confirmed that implanted bioengineered sphincters maintain IAS physio-

In summary, IAS sphincters using smooth muscle tissue could be bioengineered.

**model**

C57BL/ 6J mice

C57BL/ 6J mice

C57BL/ 6J mice

C57BL/ 6J mice model

**Key outcome (e.g., safety (tumor/tox/biodistribution), efficacy, characterization, stability, degradation)**

Smooth muscle cells expressed cell lineage appropriate phenotype markers

Formation of stable sphincters

Bioengineered sphincters were implanted subcutaneously on syngeneic mice (C57BL/6J)

No fibrosis or inflammation was observed in bioengineered sphincter implants

The bioengineered sphincters were cytocompatibility, functional, without any adverse reaction and had potential to be used as a graft for dysfunctional internal

> In vitro expanded IAS smooth muscle cells

Bioengineered sphincters

Bioengineered sphincters

Implanted bioengineered sphincters

*Summary of nonclinical study for safety and efficacy of bioengineered sphincters in C57BL/6J mice.*

maintained smooth muscle alignment and phenotype [37, 38].

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

logical functionality after implantation [37, 38].

**Steps Study objective(s) Test article Animal**

To isolate IAS smooth muscle cells (SMC) and characterization of smooth muscle

Characterize the bioengineered sphincters

Optimization of the implantation procedure

Analysis of fibrosis/ inflammation and functional activity

*Study purpose: implantation of bioengineered sphincters into rodent*

anal sphincter [37, 38].

*Study purpose: cell isolation*

*Study purpose: bioengineered sphincters*

*Study purpose: end points analysis*

Isolation of SMC

Bioengineering sphincters with smooth muscle

Implantation of bioengineered sphincters

Bioengineered sphincters histopathology

**Table 1.**

**103**

This chapter encompasses both in vitro and in vivo studies designed to support the safety and efficacy of bioengineered sphincters. Studies performed in vitro include the generation of three-dimensional internal anal sphincter models using rabbit IAS smooth muscle cells and human IAS smooth muscle cells. The in vitro studies also describe the intrinsical innervation of bioengineered IAS sphincters. Studies performed in vivo are described in two parts, small animal rodent studies and a large animal, rabbit fecal incontinent model. Small animal rodent studies included: (1) generation and implantation of IAS smooth muscle cell sphincter into a C57BL/6 J rodent; (2) generation and implantation of human innervated bioengineered sphincters into an athymic rodent model, at subcutaneous and peri-anal sites. Large animal studies demonstrating successful implantation of intrinsically innervated autologous IAS BioSphincters were conducted in a rabbit model of fecal incontinent.

#### **2. Bioengineering an in vitro three-dimensional physiological model of the internal anal sphincter from rabbit smooth muscle cells**

The objective of the early studies was to develop an in-vitro three-dimensional (3-D) physiological model of the IAS smooth muscle cells. In this initial attempt, rabbit origin IAS smooth muscles were cultured on top of a loose fibrin gel; subsequently, these cells migrated and self-assembled in circumferential alignment. As the cells matured, the fibrin gel contracted around a 5-mm-diameter silicon mold, resulting in a 3-D cylindrical ring of sphincteric tissue [40].

Histological analysis exhibited a gradient of cell alignment in the bioengineered IAS sphincters. The engineered sphincters were analyzed for physiological functionality using an isometric force transducer. Constructs were placed between a stationary central pin and the measuring arm of the organ bath transducer (Harvard Apparatus, Holliston, MA). The bioengineered sphincter generated a spontaneous basal tone, and treatment with 8-bromo-cAMP (8-Br-cAMP) resulted in relaxation. In the next step, agonist-induced stimulation (using acetylcholine) resulted in calcium- and concentration-dependent peak contraction. This effect was diminished by the addition of 8-Br-cAMP. Similar bioengineered IAS sphincters were also generated using colonic smooth muscle cells. IAS constructs display significant differences in functionality compared to colonic smooth muscle cells constructs, which confirmed tissue specificity and functionally to IAS [40].

This was the first successful attempt to develop 3-D in vitro model of engineered IAS sphincters using smooth muscle cells of IAS. Bioengineered IAS sphincters displayed circular cell alignment and physiological functionality. The functionality and physiological response in engineered tissues exhibited similarity to IAS smooth muscle in vivo [38].

#### **3. In vivo cytocompatibility and functionality analysis on subcutaneous implantation of physiologically functional bioengineered internal anal sphincter**

After successful bioengineering an IAS specific sphincter tissues, the next goal was to evaluate the in vivo biocompatibility and adverse reaction. The objective of these studies was to test the post-implantation functionality of bioengineered sphincters engineered using IAS smooth muscle cells. **Table 1** summarized the detail study design.

#### *BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*

This chapter summarizes the regenerative medicine approach of bioengineering of BioSphincters, including developmental stages of the technology, challenges, process optimization, characterization, detail pre-clinical evaluation of the

This chapter encompasses both in vitro and in vivo studies designed to support the safety and efficacy of bioengineered sphincters. Studies performed in vitro include the generation of three-dimensional internal anal sphincter models using rabbit IAS smooth muscle cells and human IAS smooth muscle cells. The in vitro studies also describe the intrinsical innervation of bioengineered IAS sphincters. Studies

performed in vivo are described in two parts, small animal rodent studies and a large animal, rabbit fecal incontinent model. Small animal rodent studies included: (1) generation and implantation of IAS smooth muscle cell sphincter into a C57BL/6 J rodent; (2) generation and implantation of human innervated bioengineered sphincters into an athymic rodent model, at subcutaneous and peri-anal sites. Large animal studies demonstrating successful implantation of intrinsically innervated autologous

IAS BioSphincters were conducted in a rabbit model of fecal incontinent.

resulting in a 3-D cylindrical ring of sphincteric tissue [40].

which confirmed tissue specificity and functionally to IAS [40].

muscle in vivo [38].

**anal sphincter**

**102**

**2. Bioengineering an in vitro three-dimensional physiological model of the internal anal sphincter from rabbit smooth muscle cells**

The objective of the early studies was to develop an in-vitro three-dimensional (3-D) physiological model of the IAS smooth muscle cells. In this initial attempt, rabbit origin IAS smooth muscles were cultured on top of a loose fibrin gel; subsequently, these cells migrated and self-assembled in circumferential alignment. As the cells matured, the fibrin gel contracted around a 5-mm-diameter silicon mold,

Histological analysis exhibited a gradient of cell alignment in the bioengineered IAS sphincters. The engineered sphincters were analyzed for physiological functionality using an isometric force transducer. Constructs were placed between a stationary central pin and the measuring arm of the organ bath transducer (Harvard Apparatus, Holliston, MA). The bioengineered sphincter generated a spontaneous basal tone, and treatment with 8-bromo-cAMP (8-Br-cAMP) resulted in relaxation. In the next step, agonist-induced stimulation (using acetylcholine) resulted in calcium- and concentration-dependent peak contraction. This effect was diminished by the addition of 8-Br-cAMP. Similar bioengineered IAS sphincters were also generated using colonic smooth muscle cells. IAS constructs display significant differences in functionality compared to colonic smooth muscle cells constructs,

This was the first successful attempt to develop 3-D in vitro model of engineered

**3. In vivo cytocompatibility and functionality analysis on subcutaneous implantation of physiologically functional bioengineered internal**

After successful bioengineering an IAS specific sphincter tissues, the next goal was to evaluate the in vivo biocompatibility and adverse reaction. The objective of these studies was to test the post-implantation functionality of bioengineered sphincters engineered using IAS smooth muscle cells. **Table 1** summarized the detail study design.

IAS sphincters using smooth muscle cells of IAS. Bioengineered IAS sphincters displayed circular cell alignment and physiological functionality. The functionality and physiological response in engineered tissues exhibited similarity to IAS smooth

BioSphincter towards the treatment of FI.

*Current Topics in Faecal Incontinence*

In this endeavor, smooth muscle cells were isolated from the IAS of donor C57BL/6 mice. Smooth muscle cell constructs were engineered on Sylgard coated plates using fibrin gel, as described previously [40]. The engineered constructs were successfully implanted into the subcutaneous region of same strain mice and treated with either fibroblastic growth factor-2 or saline as controls using a microosmotic pump. Mice were euthanized after 4 weeks, and the implant was harvested. The implant was intact, healthy in color without any degradation, and interestingly displayed muscle attachment to the back of the mouse, with neovascularization. Constructs exhibited no external sign of inflammation, fibrosis, or infection, because of the use of syngeneic tissue. The supplement of FGF-2 also helped in tissue viability, cellular integrity, and vascularization. The harvested tissues maintained smooth muscle alignment and phenotype [37, 38].

The post-implant harvested constructs were analyzed for force generation. The harvested implants generated and maintained the spontaneous basal tone in the absence of any external stimuli. The developed tone confirmed the integrity of ionic membrane characteristics, membrane receptors and their intracellular signaling mechanisms for contraction and relaxation. On treatment of a relaxing stimulant such as a vasoactive intestinal peptide (VIP), the force and magnitude of relaxation were consistent before and after implantation. The rapid, and dose-dependent sustained (over 30 min without signs of muscle fatigue) contractions on the treatment of acetylcholine and phorbol dibutyrate was elicited as well. The physiological studies confirmed that implanted bioengineered sphincters maintain IAS physiological functionality after implantation [37, 38].

In summary, IAS sphincters using smooth muscle tissue could be bioengineered. The bioengineered sphincters were cytocompatibility, functional, without any adverse reaction and had potential to be used as a graft for dysfunctional internal anal sphincter [37, 38].


#### **Table 1.**

*Summary of nonclinical study for safety and efficacy of bioengineered sphincters in C57BL/6J mice.*

#### **4. Bioengineering an internal anal sphincter derived from smooth muscle cells isolated from the human internal anal sphincter**

The preliminary work in the previous sections using SMCs harvested from animal models confirmed the feasibility of engineering functional physiologic IAS constructs and initial biocompatibility. [40]. The next objective was to validate the feasibility of engineering IAS sphincter constructs from SMCs of human IAS origin.

Human IAS was received from NDRI and SMCs were harvested following previously described protocol. At confluency, SMCs were seeded on Sylgard coated plates with fibrin gel. Cells migrated and aligned circularly around the Sylgard mold located at the center of the plate. All the 3-D bioengineered sphincter constructs successfully formed within 5–10 days of seeding of Human IAS SMCs [34].

The developed human IAS constructs displayed the essential characteristics of a native functional IAS; the bioengineered IAS constructs able to generate the spontaneous myogenic basal tone and respond to different pharmacological agents. Bioengineered human IAS sphincters also exhibited dose-dependent force generation in response to different stimulants. The IAS smooth muscle constructs displayed a tissue-specific basal tone compared to colonic muscle cells. The basal tone, acetylcholine-induced contraction and PdBU generated were reduced by calphostin-C but not with Y-27632. The detailed functionality resulted that the protein kinase C (PKC) pathway (independent of the Rho/ROCK pathway) appeared to be responsible for IAS specific tone and contractions [34].

The process of bioengineering IAS constructs using human IAS smooth muscles was highly reproducible. The developed IAS muscle constructs were functionally similar to native IAS sphincters. This was the first report demonstrating the generation of a functional in vitro model of human IAS that may be used for the elucidation of mechanisms associated with smooth muscle sphincter myogenic malfunction and for the investigation of treatments for fecal incontinence [34].

#### **5. Bioengineered IAS generated from human cells and preliminary biocompatibility and functional analysis after implantation in an athymic rodent model**

In the previous sections, IAS muscle constructs were successfully bioengineered with animal and human origin IAS circular muscles. The bioengineered mouse IAS muscle constructs displayed physiological functionality after implantation in wild type mice. However, compare to anatomy and physiology of native IAS sphincters, the bioengineered muscle constructs lacked innervation of the neuronal population. Therefore, the next target in these studies was to intrinsically innervation of bioengineered IAS muscle constructs and evaluation of cellular viability, physiological functionality, and safety after implantation. **Table 2** summarized the detail study design.

process took 9 days to generate an intrinsically innervated muscle constructs mimicking physiological functionality to native IAS tissues. The neural cell differentiation was further confirmed by positive expression of mature excitatory (choline acetyltransferase; ChAT) and inhibitory (VIP) motor neurons in the quantitative analysis using PCR. The cross-sections of engineered sphincters were demonstrated positive immunoreactivity against ChAT and VIP markers. After physiological functional analysis, the bioengineered sphincter were implanted subcutaneously

*Summary of nonclinical study for safety and efficacy of bioengineered sphincters in athymic rodent model.*

**Test article Animal model Key outcome (e.g., safety**

Cadaver human

h-2kb-tsA58 immortomouse

Cadaver human and h-2kb-tsA58 immortomouse

RAG1/

**(tumor/tox/**

**degradation)**

markers

RAG1/mice Bioengineered sphincters were implanted

mice • No fibrosis or

**biodistribution), efficacy, characterization, stability,**

Smooth muscle cells expressed cell lineage appropriate phenotype markers

Neural cells expressed cell lineage appropriate phenotype

• Formation of

sphincters • Bioengineered sphincters exhibited basal tone, relaxation and contractile activity

subcutaneously on athymic mice (RAG1/) model

> inflammation was observed in Bioengineered Sphincter implants • Harvested post-implant sphincters capable of maintaining basal tone, relaxation, and contractile activity

intrinsically innervated

At harvest, the implanted construct exhibited neo-vascularization without any symptom of fibrosis or immunogenic reaction. The immuno-histological analysis confirmed that the sections of the harvested implant displayed reticulated

into immune suppressed RAG1/ mice for 4 weeks [35].

**Steps Study objective (s)**

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

*Study purpose: bioengineered sphincters*

smooth muscle cells (SMC) and characterization

*BioSphincter a Regenerative Medicine Approach to Treat FI*

To isolate neural cells and characterization

Characterize the bioengineered sphincters

*Study purpose: implantation of bioengineered sphincters into rodent*

Optimization of the implantation procedure

Analysis of fibrosis/ inflammation and functional activity

In vitro expanded IAS smooth muscle cells

In vitro expanded neural cells

Bioengineered sphincters

Bioengineered sphincters

Implanted bioengineered sphincters

*Study purpose: cell isolation* Isolation of SMC To isolate IAS

Isolation of neural cells from embryo of immortomouse

Bioengineered Sphincters with smooth muscle and neural cells

Implantation of bioengineered sphincters

Bioengineered sphincters histopathology

**Table 2.**

**105**

*Study purpose: end points analysis*

In this effort, the human IAS muscles were harvested and cultured as described previously. The neuronal cell line was isolated from a D13 embryo from H-2KbtsA58 immortomouse. The bioengineering of constructs was divided into two steps. In the first step, the isolated neuronal stem cells were mixed with hydrogel and plated in the Sylgard coated plates. After gelation, IAS origin smooth muscle cells were mixed with the collagen gel and overlaid to the previous cell-hydrogel. A fully compacted sphincter-like construct were developed in the first 60 h [35].

The neuronal stem cells differentiation towards functional neurons was carried out in a specific media targeted to neural differentiation. The bioengineering


#### **Table 2.**

**4. Bioengineering an internal anal sphincter derived from smooth muscle cells isolated from the human internal anal sphincter**

human IAS origin.

*Current Topics in Faecal Incontinence*

IAS SMCs [34].

The preliminary work in the previous sections using SMCs harvested from animal models confirmed the feasibility of engineering functional physiologic IAS constructs and initial biocompatibility. [40]. The next objective was to validate the feasibility of engineering IAS sphincter constructs from SMCs of

Human IAS was received from NDRI and SMCs were harvested following previously described protocol. At confluency, SMCs were seeded on Sylgard coated plates with fibrin gel. Cells migrated and aligned circularly around the Sylgard mold located at the center of the plate. All the 3-D bioengineered sphincter constructs successfully formed within 5–10 days of seeding of Human

The developed human IAS constructs displayed the essential characteristics of a native functional IAS; the bioengineered IAS constructs able to generate the spontaneous myogenic basal tone and respond to different pharmacological agents. Bioengineered human IAS sphincters also exhibited dose-dependent force generation in response to different stimulants. The IAS smooth muscle constructs displayed a tissue-specific basal tone compared to colonic muscle cells. The basal tone, acetylcholine-induced contraction and PdBU generated were reduced by calphostin-C but not with Y-27632. The detailed functionality resulted that the protein kinase C (PKC) pathway (independent of the Rho/ROCK pathway) appeared to be responsible for IAS specific tone and contractions [34].

The process of bioengineering IAS constructs using human IAS smooth muscles was highly reproducible. The developed IAS muscle constructs were functionally similar to native IAS sphincters. This was the first report demonstrating the generation of a functional in vitro model of human IAS that may be used for the elucida-

tion of mechanisms associated with smooth muscle sphincter myogenic

and safety after implantation. **Table 2** summarized the detail study design.

compacted sphincter-like construct were developed in the first 60 h [35].

out in a specific media targeted to neural differentiation. The bioengineering

**in an athymic rodent model**

**104**

malfunction and for the investigation of treatments for fecal incontinence [34].

**5. Bioengineered IAS generated from human cells and preliminary biocompatibility and functional analysis after implantation**

In the previous sections, IAS muscle constructs were successfully bioengineered with animal and human origin IAS circular muscles. The bioengineered mouse IAS muscle constructs displayed physiological functionality after implantation in wild type mice. However, compare to anatomy and physiology of native IAS sphincters, the bioengineered muscle constructs lacked innervation of the neuronal population. Therefore, the next target in these studies was to intrinsically innervation of bioengineered IAS muscle constructs and evaluation of cellular viability, physiological functionality,

In this effort, the human IAS muscles were harvested and cultured as described previously. The neuronal cell line was isolated from a D13 embryo from H-2KbtsA58 immortomouse. The bioengineering of constructs was divided into two steps. In the first step, the isolated neuronal stem cells were mixed with hydrogel and plated in the Sylgard coated plates. After gelation, IAS origin smooth muscle cells were mixed with the collagen gel and overlaid to the previous cell-hydrogel. A fully

The neuronal stem cells differentiation towards functional neurons was carried

*Summary of nonclinical study for safety and efficacy of bioengineered sphincters in athymic rodent model.*

process took 9 days to generate an intrinsically innervated muscle constructs mimicking physiological functionality to native IAS tissues. The neural cell differentiation was further confirmed by positive expression of mature excitatory (choline acetyltransferase; ChAT) and inhibitory (VIP) motor neurons in the quantitative analysis using PCR. The cross-sections of engineered sphincters were demonstrated positive immunoreactivity against ChAT and VIP markers. After physiological functional analysis, the bioengineered sphincter were implanted subcutaneously into immune suppressed RAG1/ mice for 4 weeks [35].

At harvest, the implanted construct exhibited neo-vascularization without any symptom of fibrosis or immunogenic reaction. The immuno-histological analysis confirmed that the sections of the harvested implant displayed reticulated

neural network innervated into intact aligned muscles. The section displayed microvasculature and several blood vessels embedded within the implanted smooth muscles [35].

The constructs responded appropriately to physiologically relevant stimulatory and inhibitory neurotransmitters during functional analysis. It was validated in immunocytochemistry, the intrinsically innervated bioengineered construct exhibited excitatory and inhibitory motor neuronal population. The constructs displayed characteristics of functional mature contractile IAS smooth muscle as well. Overall, the human innervated functional IAS sphincter like tissues were

**7. Peri-anal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural**

After successful bioengineering of human IAS sphincter-like tissues, it was essential to evaluate the in vivo safety and functionality. In the next part of the study, a method was developed for isolation of rectal verge in an athymic rodent

**Test article Animal**

In vitro expanded IAS smooth muscle cells

In vitro expanded neural progenitor cells

Bioengineered sphincters

Bioengineered sphincters

Implanted bioengineered sphincters

**model**

Cadaver human

Cadaver human

Cadaver

athymic nude rats

athymic nude rats

*Summary of nonclinical study of safety and efficacy of peri-anal implantation of human origin bioengineered*

**Key outcome (e.g., safety (tumor/tox/**

Smooth muscle cells expressed cell lineage appropriate phenotype markers

Neural progenitor cells expressed cell lineage appropriate phenotype markers

innervated sphincters • Bioengineered sphincters exhibited basal tone, relaxation, and contractile activity

Bioengineered sphincters were implanted peri-anal site on athymic

> • No fibrosis or inflammation was observed in Bioengineered

Sphincter implants • Harvested post-implant sphincters capable of maintaining basal tone, relaxation, and contractile

activity

nude rats model

human • Formation of intrinsically

**biodistribution), efficacy, characterization, stability,**

**degradation)**

successfully bioengineered and characterized [33].

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

**progenitor cells**

**Steps Study objective (s)**

Isolation of SMC To isolate IAS

smooth muscle cells (SMC) and characterization

To isolate neural progenitor cells

characterization

Characterize the bioengineered sphincters

*Study purpose: implantation of bioengineered sphincters into rodent*

Optimization of the implantation procedure

Analysis of fibrosis/ inflammation and functional activity

and

*Study purpose: bioengineered sphincters*

*Study purpose: cell isolation*

Isolation of neural progenitor cells

Bioengineered sphincters with smooth muscle and neural progenitor

Implantation of bioengineered sphincters

Bioengineered sphincters histopathology

**Table 3.**

**107**

*Study purpose: end points analysis*

*sphincters into athymic rodent model.*

cells

The myogenic and neuronal components were preserved after implantation. All the bioengineered constructs were able to generate myogenic spontaneous basal tone pre- and post-implantation. A rapid and robust relaxation response was observed against VIP. This relaxation was 50–70% attenuated on pre-treatment of TTX, indicated that VIP-induced relaxation has both neuronal, as well as myogenic component. The relaxation was further validated with EFS and resulted in transient relaxation ultimately recovered to basal tone. The inhibition of nitrergic and VIPergic EFS-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction) confirmed the relaxation of enteric nerves results in nitrergic as well as VIP-ergic inhibitory neurotransmission in the implants. The excitatory neurotransmitter Ach (and partial inhibition on pre-treatment with TTX)-induced contraction response emulated before and after implantation, confirmed synergistic involvement of both neuronal and myogenic components. Fundamental electromechanical coupling of smooth muscle was also maintained during implantation, rendering the implanted IAS physiologically similar to in vivo IAS [35].

This was the first attempt of bioengineering of intrinsically innervated human IAS constructs. Both of myogenic and neuronal components of constructs were stable, sustained, viable and synergistically responsive after implantation in immune-suppressed mice. The study also concluded that bioengineering of intrinsically innervated sphincter is feasible, scalable, and customizable to match specific size and cell population. This leads to one step closer towards bioengineering of human engineered BioSphincters.

#### **6. Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs**

In previous studies, IAS smooth muscle constructs were engineered [34, 40] and implanted for cytocompatibility and physiological analysis. These preliminary studies were proof of concept using human origin SMCs and immortomouse-origin neural stem cells. To translate the bioengineered sphincter to the clinical realm, it was essential to use human origin neural cells to engineer IAS sphincters.

The next objective was to develop bioengineering physiologically functional, intrinsically innervated human IAS tissues, using human origin neural cells and IAS muscle cells. Therefore, a method was optimized for the isolation of neuronal progenitor cells (NPCs) from intestinal biopsies of adult human donors. The cell culture and characterization protocol were standardized to yield an undifferentiated pure population of enteric neural progenitor cells [33].

Several matrix compositions were evaluated as a carrier for differentiation of adult enteric NPCs to functional neurons. The type-1 collagen with laminin was optimized as hydrogel for neural differentiation [41, 42]. The collagen acts as a matrix for mechanical strength and laminin is important for neuronal development. The SMCs has the ability to reform the collagen hydrogel into 3D structure due to matrix metalloproteinase activity [43]. During this restructure of hydrogel from 2D to 3D, SMCs came into close proximities of NPCs and enhanced the NPCs differentiation. Detail NPCs-SMCs interactions were studied, and it was observed that mature smooth muscle was essential for the direct differentiation of adult enteric NPCs [33]. The ratio of NPCs and SMCs were also studied and concluded that 200,000 NPCs/construct with 500,000 SMC/constructs were optimum do generate a native physiological response [33].

*BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*

neural network innervated into intact aligned muscles. The section displayed microvasculature and several blood vessels embedded within the implanted

implanted IAS physiologically similar to in vivo IAS [35].

**human internal anal sphincter constructs**

human engineered BioSphincters.

a native physiological response [33].

**106**

The myogenic and neuronal components were preserved after implantation. All the bioengineered constructs were able to generate myogenic spontaneous basal tone pre- and post-implantation. A rapid and robust relaxation response was observed against VIP. This relaxation was 50–70% attenuated on pre-treatment of TTX, indicated that VIP-induced relaxation has both neuronal, as well as myogenic component. The relaxation was further validated with EFS and resulted in transient relaxation ultimately recovered to basal tone. The inhibition of nitrergic and VIPergic EFS-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction) confirmed the relaxation of enteric nerves results in nitrergic as well as VIP-ergic inhibitory neurotransmission in the implants. The excitatory neurotransmitter Ach (and partial inhibition on pre-treatment with TTX)-induced contraction response emulated before and after implantation, confirmed synergistic involvement of both neuronal and myogenic components. Fundamental electromechanical coupling of smooth muscle was also maintained during implantation, rendering the

This was the first attempt of bioengineering of intrinsically innervated human IAS constructs. Both of myogenic and neuronal components of constructs were stable, sustained, viable and synergistically responsive after implantation in immune-suppressed mice. The study also concluded that bioengineering of intrinsically innervated sphincter is feasible, scalable, and customizable to match specific size and cell population. This leads to one step closer towards bioengineering of

**6. Bioengineering of physiologically functional intrinsically innervated**

implanted for cytocompatibility and physiological analysis. These preliminary studies were proof of concept using human origin SMCs and immortomouse-origin neural stem cells. To translate the bioengineered sphincter to the clinical realm, it

was essential to use human origin neural cells to engineer IAS sphincters.

culture and characterization protocol were standardized to yield an undifferentiated pure population of enteric neural progenitor cells [33].

In previous studies, IAS smooth muscle constructs were engineered [34, 40] and

The next objective was to develop bioengineering physiologically functional, intrinsically innervated human IAS tissues, using human origin neural cells and IAS muscle cells. Therefore, a method was optimized for the isolation of neuronal progenitor cells (NPCs) from intestinal biopsies of adult human donors. The cell

Several matrix compositions were evaluated as a carrier for differentiation of adult enteric NPCs to functional neurons. The type-1 collagen with laminin was optimized as hydrogel for neural differentiation [41, 42]. The collagen acts as a matrix for mechanical strength and laminin is important for neuronal development. The SMCs has the ability to reform the collagen hydrogel into 3D structure due to matrix metalloproteinase activity [43]. During this restructure of hydrogel from 2D to 3D, SMCs came into close proximities of NPCs and enhanced the NPCs differentiation. Detail NPCs-SMCs interactions were studied, and it was observed that mature smooth muscle was essential for the direct differentiation of adult enteric NPCs [33]. The ratio of NPCs and SMCs were also studied and concluded that 200,000 NPCs/construct with 500,000 SMC/constructs were optimum do generate

smooth muscles [35].

*Current Topics in Faecal Incontinence*

The constructs responded appropriately to physiologically relevant stimulatory and inhibitory neurotransmitters during functional analysis. It was validated in immunocytochemistry, the intrinsically innervated bioengineered construct exhibited excitatory and inhibitory motor neuronal population. The constructs displayed characteristics of functional mature contractile IAS smooth muscle as well. Overall, the human innervated functional IAS sphincter like tissues were successfully bioengineered and characterized [33].

#### **7. Peri-anal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells**

After successful bioengineering of human IAS sphincter-like tissues, it was essential to evaluate the in vivo safety and functionality. In the next part of the study, a method was developed for isolation of rectal verge in an athymic rodent


#### **Table 3.**

*Summary of nonclinical study of safety and efficacy of peri-anal implantation of human origin bioengineered sphincters into athymic rodent model.*

model. Athymic nude mice were larger animal compared to normal mice. The selection of immune deficient rat for implantation studies of human-origin bioengineered constructs was to avoid any immune rejection.

**Steps Study objective(s) Test article Animal**

To induce FI Donor IAS

tissue

In vitro expanded IAS smooth muscle cells

In vitro expanded small intestine neural progenitor cells

Autologous bioengineered sphincters

Autologous bioengineered sphincters

Autologous bioengineered sphincters

Implanted bioengineered sphincters

Implanted bioengineered sphincters

*Study purpose: implantation of engineered autologous bioengineered sphincters to treat FI in rabbits*

*Study purpose: developing FI model and autologous cell isolation*

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

IAS smooth muscle cells (SMC) characterization of autologous smooth

Isolation of SMC To isolate autologous

muscle

*Study purpose: autologous bioengineered sphincters*

To isolate neural progenitor cells (NPC) characterization of autologous NPC

Characterize the bioengineered sphincters

Optimization of the implantation procedure

Effects of bioengineered sphincters on the restoration of continence

IAS hemisphincterectomy

Small intestinal biopsy

Bioengineered sphincters with autologous smooth muscle and neural progenitor cells

Implantation of bioengineered sphincters

Anal basal pressure and RAIR

*Study purpose: end points analysis*

**109**

Blood results Effects of implants on

Tissue pathology Effects of experimental

blood cell counts, kidney and liver function, and electrolytes.

conditions on tissue pathology

**model**

Female New Zealand rabbits

Female New Zealand rabbits

female New Zealand rabbits

female New Zealand rabbits

female New Zealand rabbits

female New Zealand rabbits

female New Zealand rabbits

female New Zealand rabbits

**Key outcome (e.g., safety**

Lack of fecal hygiene and significant reduction in anal basal pressure and

Smooth muscle cells expressed cell lineage appropriate phenotype

Neural progenitor cells expressed cell lineage appropriate phenotype

Restoration of fecal hygiene, anal basal pressure, and RAIR

The dosage of

bioengineered sphincters was optimized four bioengineered sphincters were implanted on each rabbit in the treated group

Rabbits with induced FI receiving bioengineered sphincter implants had anal basal pressure, and RAIR restored to normal baseline, but rabbits with induced FI in the nontreated group and sham surgery group had consistently reduced anal basal pressure and RAIR

There were no adverse effects of implants on blood values

There were no effects of experimental condition on local or peripheral histopathology

**(tumor/tox/ biodistribution), efficacy, characterization, stability, degradation)**

RAIR

markers

markers

The intrinsically innervated human IAS Sphincter were bioengineered using IAS origin SMCs and enteric NPCs. The developed surgical models were used to implant bioengineered sphincter into the perianal region of athymic rats for 4 weeks, following assessment of viability and functionality [36]. All the rats survived till respective time points without any obstruction or difficulty with defecation or fecal accumulation. Histopathology analysis concluded the absence of any abscess formations, infection, or adverse reaction. The implanted constructs were stable and intact at perirectal tissue of the rat, without any sign of fibrosis or neoplasia. Immuno-histological analysis with endothelial-specific antigen, von Willebrand's factor confirmed neovascularization and formation of several blood vessels. The contractile smooth muscle phenotype was maintained by exhibiting positive expression to human reactive muscle specific antibodies. **Table 3** summarized the detail study design [36].

Pre- and post-implant physiological force measurement studies confirmed distinct characteristics like native sphincters. The engineered IAS sphincter exhibited stable spontaneous myogenic basal tone. There was a robust response to different relaxant and excitatory stimulants, which was persistent after implantation.

This study concluded that for clinical application the bioengineered sphincter could be used in an additive manner rather than in a replacement manner, where native compromised IAS sphincter can be supported by transplantation of additional bioengineered sphincters. In this way, the patient's own IAS can be preserved and augmented with additional autologous functional neuro-muscular components [36].

#### **8. Long-term non-clinical study of autologous bioengineered BioSphincters for the treatment of fecal incontinence**

This study aimed to provide data for a large animal model in support of the use of Bioengineered sphincter as a new therapy to treat FI. These nonclinical studies were conducted to test the safety and efficacy of using autologous cell bioengineered sphincters as a regenerative medicine approach for treating induced FI in rabbits. The study design consisted of four steps. **Table 4** summarizes the four steps including their objectives and key outcomes.

#### **8.1 Selection of a large animal model for nonclinical studies of fecal incontinence**

Currently, there is no model for FI where the defect is specific to the internal anal sphincter. In humans, the IAS is responsible for 70% of anal basal pressure, anal closure, and fecal continence. The New Zealand white rabbit (female, 3.0– 3.5 kg at the enrollment of the study) was chosen as an animal model because the anatomy and the surgical planes of the anal area are similar to humans. The rabbit was selected as a good model for successful identification and surgical resection of full thickness biopsies with a successful outcome. Thus, the rabbit is a good large animal model for our lab to utilize in evaluating FI. The number of animals, experimental protocols, and overall study design used in this study were reviewed and approved by the Wake Forest Institutional Animal Care and Use Committee before conducting any component of this study involving animals. Each rabbit was given a unique identification number that was printed on the cage card. Each rabbit was identified using a unique identification number. All data collected on each animal was referenced with the unique animal identification number and tattooed onto the model. Athymic nude mice were larger animal compared to normal mice. The selection of immune deficient rat for implantation studies of human-origin

cle specific antibodies. **Table 3** summarized the detail study design [36].

**8. Long-term non-clinical study of autologous bioengineered BioSphincters for the treatment of fecal incontinence**

were conducted to test the safety and efficacy of using autologous cell

**8.1 Selection of a large animal model for nonclinical studies of fecal**

steps including their objectives and key outcomes.

**incontinence**

**108**

The intrinsically innervated human IAS Sphincter were bioengineered using IAS origin SMCs and enteric NPCs. The developed surgical models were used to implant bioengineered sphincter into the perianal region of athymic rats for 4 weeks, following assessment of viability and functionality [36]. All the rats survived till respective time points without any obstruction or difficulty with defecation or fecal accumulation. Histopathology analysis concluded the absence of any abscess formations, infection, or adverse reaction. The implanted constructs were stable and intact at perirectal tissue of the rat, without any sign of fibrosis or neoplasia. Immuno-histological analysis with endothelial-specific antigen, von Willebrand's factor confirmed neovascularization and formation of several blood vessels. The contractile smooth muscle phenotype was maintained by exhibiting positive expression to human reactive mus-

Pre- and post-implant physiological force measurement studies confirmed distinct characteristics like native sphincters. The engineered IAS sphincter exhibited stable spontaneous myogenic basal tone. There was a robust response to different relaxant and excitatory stimulants, which was persistent after implantation. This study concluded that for clinical application the bioengineered sphincter could be used in an additive manner rather than in a replacement manner, where native compromised IAS sphincter can be supported by transplantation of additional bioengineered sphincters. In this way, the patient's own IAS can be preserved and augmented with additional autologous functional neuro-muscular components [36].

This study aimed to provide data for a large animal model in support of the use of Bioengineered sphincter as a new therapy to treat FI. These nonclinical studies

bioengineered sphincters as a regenerative medicine approach for treating induced FI in rabbits. The study design consisted of four steps. **Table 4** summarizes the four

Currently, there is no model for FI where the defect is specific to the internal anal sphincter. In humans, the IAS is responsible for 70% of anal basal pressure, anal closure, and fecal continence. The New Zealand white rabbit (female, 3.0– 3.5 kg at the enrollment of the study) was chosen as an animal model because the anatomy and the surgical planes of the anal area are similar to humans. The rabbit was selected as a good model for successful identification and surgical resection of full thickness biopsies with a successful outcome. Thus, the rabbit is a good large animal model for our lab to utilize in evaluating FI. The number of animals, experimental protocols, and overall study design used in this study were reviewed and approved by the Wake Forest Institutional Animal Care and Use Committee before conducting any component of this study involving animals. Each rabbit was given a unique identification number that was printed on the cage card. Each rabbit was identified using a unique identification number. All data collected on each animal was referenced with the unique animal identification number and tattooed onto the

bioengineered constructs was to avoid any immune rejection.

*Current Topics in Faecal Incontinence*



#### **Table 4.**

*Summary of nonclinical study of safety and efficacy of bioengineered sphincters.*

ear of each animal to prevent mix-up. Rabbits were acclimated for at least 6 days before enrollment in the study [10, 39].

#### **8.2 Study groups**

The groups of the study, summarized in **Table 5**, was developed to assess the postimplantation safety of bioengineered sphincters in rabbits at three-time points (3, 6, and 12 months). All animals underwent IAS hemi-sphincterectomy to induce FI. Rabbits were randomly divided into three experimental groups: (1) non-treated group (incontinent control), (2) treated group (received surgical implantation of bioengineered sphincters 6–8 weeks following sphincterectomy through a surgical opening of the anal verge), and (3) Sham surgery group (surgical opening of the anal verge was performed followed by immediate closure without implantation of bioengineered sphincters).

#### *8.2.1 Development of FI*

The IAS hemi-sphincterectomy was performed on all the rabbits to induce passive FI. The development of passive FI was confirmed in each assessment of fecal hygiene and anorectal pressure. Baseline manometry readings were obtained on all rabbits before any surgeries. Following hemi-sphincterectomy, anorectal manometry was performed on all rabbits to confirm passive FI, which was identified by lack of fecal hygiene and by a significant decrease in anal basal pressure and RAIR in all rabbits [10, 39].

#### *8.2.2 Bioengineering of autologous BioSphincters*

The SMCs were isolated from the IAS harvested during hemi-sphincterectomy. Isolated cells were characterized by α-smooth muscle actin and smoothelin markers. Cells stained positive confirming contractile phenotype of smooth muscle cells. NPCs were isolated from small intestine biopsies. Cells were then characterized by immunofluorescence and stained positive for p75NTR, Nestin, and Sox2, confirming neural crest-derived stem cells. Both cell types were expanded for 4 weeks to obtain the required number to form the bioengineered sphincters.

Intrinsically innervated IAS sphincters were bioengineered using both types of cells as described previously. Bioengineered sphincter products were characterized using different methods. The presence of aligned smooth muscle cells and the

**Study groups (no. of**

**111**

**Baseline**

**Sphincterectomy**

**induce FI**

 **to**

**Manometry**

 **post**

**4–6 weeks post**

**1 month 3 months 6 months 12 months**

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

**sphincterectomy**

No treatment

Implant sphincters

Sham surgery

Manometry

 post sham

bioengineered

Manometry

 post implant

Manometry

 post

sphincterectomy

**sphincterectomy**

 ✓

 ✓

 ✓

**manometry**

✓✓

✓✓

**rabbits)**

Non-treated

Treated group (10)

Sham surgery group (5)

**Table 5.** *Study groups for the non-clinical*

 *study.*

✓✓

 group (11)

#### *BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*


**Table 5.** *Study*

 *groups for the non-clinical study.*

ear of each animal to prevent mix-up. Rabbits were acclimated for at least 6 days

bioengineered sphincters

bioengineered sphincters bioengineered sphincter

**model**

female New Zealand rabbits

female New Zealand rabbits

**Key outcome (e.g., safety**

There were no effects of bioengineered sphincter implantation on morbidity

No definitive difference between bioengineered sphincter implants and naïve. No evidence of

**(tumor/tox/ biodistribution), efficacy, characterization, stability, degradation)**

or mortality

neoplasia

**Steps Study objective(s) Test article Animal**

*Summary of nonclinical study of safety and efficacy of bioengineered sphincters.*

Morbidity/mortality Implanted

The groups of the study, summarized in **Table 5**, was developed to assess the postimplantation safety of bioengineered sphincters in rabbits at three-time points (3, 6, and 12 months). All animals underwent IAS hemi-sphincterectomy to induce FI. Rabbits were randomly divided into three experimental groups: (1) non-treated group (incontinent control), (2) treated group (received surgical implantation of bioengineered sphincters 6–8 weeks following sphincterectomy through a surgical opening of the anal verge), and (3) Sham surgery group (surgical opening of the anal verge was performed followed by immediate closure without implantation of bioengineered sphincters).

The IAS hemi-sphincterectomy was performed on all the rabbits to induce passive FI. The development of passive FI was confirmed in each assessment of fecal hygiene and anorectal pressure. Baseline manometry readings were obtained on all rabbits before any surgeries. Following hemi-sphincterectomy, anorectal manometry was performed on all rabbits to confirm passive FI, which was identified by lack of fecal hygiene and by a significant decrease in anal basal pressure and

The SMCs were isolated from the IAS harvested during hemi-sphincterectomy. Isolated cells were characterized by α-smooth muscle actin and smoothelin markers. Cells stained positive confirming contractile phenotype of smooth muscle cells. NPCs were isolated from small intestine biopsies. Cells were then characterized by

Intrinsically innervated IAS sphincters were bioengineered using both types of cells as described previously. Bioengineered sphincter products were characterized using different methods. The presence of aligned smooth muscle cells and the

immunofluorescence and stained positive for p75NTR, Nestin, and Sox2, confirming neural crest-derived stem cells. Both cell types were expanded for 4 weeks to obtain the required number to form the bioengineered sphincters.

before enrollment in the study [10, 39].

IAS histopathology Fibrosis/inflammation Implanted

**8.2 Study groups**

Clinical presentation

*Current Topics in Faecal Incontinence*

**Table 4.**

*8.2.1 Development of FI*

RAIR in all rabbits [10, 39].

**110**

*8.2.2 Bioengineering of autologous BioSphincters*

differentiated functional neural network was confirmed via immune-reactivity against smoothelin and βIII tubulin. These results further validated via positive expression of smoothelin and βIII tubulin qPCR. Engineered IAS sphincters were tested for physiological functionality. The engineered tissues able to generate the spontaneous basal tone and exhibited a robust stable response following pharmacological or electrical stimuli. The bioengineered autologous BioSphincters were implanted adjacent to IAS tissues into the respective rabbits [10, 39].

#### *8.2.3 Implantation and restoration of fecal continence*

Anorectal manometry is a technique used to measure contractility in the anus and rectum. Anorectal manometry was performed initially at baseline prior to any surgery. These measurements reflected the control state for all animals in this study. Anorectal manometry was performed prior to any surgery (before animals went for any procedure) to record the baseline, and 1 month following IAS hemisphincterectomy (biopsy), then at 3, 6, and 12 months in each experimental group.

fibrosis or avascular collagen around the implant, indicating no foreign-body reaction with the implants. Pathologic findings in this study were generally minor and consisted primarily of a low incidence of background changes and minor changes attributable to implantation. There was no evidence of neoplasia. These results confirmed that the bioengineered sphincters were viable and functional in vivo with the

*Different stages from bioengineered sphincter to implantation; (A) bioengineered sphincter; (B) implantation of two bioengineered sphincters; (C) 4 implanted bioengineered sphincters; and (D) implanted bioengineered*

In this study, passive Fi was successfully developed in the large animal model. The bioengineered intrinsically innervated IAS constructs from the autologous cells retrieved at biopsy. The IAS constructs were bioengineered and implanted after 6– 8 weeks after harvesting the cells (**Figure 2A**); then, one by one, four bioengineered sphincters were implanted at the anal site (**Figure 2B**). The four bioengineered sphincters were stacked together at the site (**Figure 2C**). After 12 months of implantation, implanted bioengineered sphincters appeared intact as one tissue at

The animals resumed normal activity and defecatory bowel movement. There

Regeneration of an intrinsically innervated function IAS sphincter is a promising

approach for long-term relief from passive FI. The IAS muscle and neural cells synergized in collagen-laminin hydrogel as a 3D sphincter like architecture, mimicking the native IAS cell orientation and innervation. The bioengineering process has been optimized, scaled up for clinical application using human origin cells. The signaling pathways for sphincter tone and contraction were characterized. The bioengineered sphincter able to generate spontaneous tone and response to different pharmacological agents was comparable to human IAS. The stability, viability and cytocompatibility analysis of engineered sphincters were carried out in vitro and in vivo conditions. The step-wise pre-clinical assessment of engineered

was no indication of any rectal outlet obstruction or anal stenosis. Anorectal manometry was performed on the animals monthly beginning 6 weeks after implantation. The animals exhibited a reinstated basal tone and RAIR. Animals were maintained and monitored up to 12 months after implantation. At each endpoint, after euthanasia, the harvested implant was tested. Results show that the construct maintained physiological functionality. The tests show that both muscle and neural type of cells maintained their physiological function. In other experiments, we have demonstrated that the cells of the implant stayed within the implant and did not

maintenance of both the muscle and neural components [10, 39].

*sphincter after euthanasia (after 12 months of implantation).*

*BioSphincter a Regenerative Medicine Approach to Treat FI*

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

the site (**Figure 2D**).

**Figure 2.**

**9. Conclusion**

**113**

migrate outside the location of the implant.

#### *8.2.3.1 Restoration of anorectal pressure*

IAS hemi-sphincterectomy resulted in a significant decrease in anal basal pressure and RAIR compared to baseline (no surgery), supporting the validity of the induced-incontinence model. In the sham surgery group, anal basal pressure and RAIR were not improved and were comparable to readings from rabbits in the nontreated group. Compared to baseline, the basal pressure in non-treated and sham group was decreased by 41% (p < 0.0001) after 1 month of hemi-sphincterectomy and remained low up to study time point of 12 months. Similarly, RAIR was also reduced by 50.9% from the baseline (p < 0.0001). It remained low in non-treated group (49.2%) and sham groups (40.0%) compared to baseline till the study time point.

This reduced anorectal functionality was restored within 1-month postimplantation of autologous BioSphincters in the treated group. The resting pressure was returned to baseline after 4 weeks of implantation and remained similar up to 12 months. RAIR was restored by 88% in initial 1 month and improved within 3 months and sustained till 12 months. The restoration of basal pressure and RAIR were significantly higher (p < 0.0001) than values observed in the non-treated group and sham groups.

#### *8.2.3.2 Improvement in fecal hygiene*

The IAS hemi-sphincterectomy affected fecal hygiene of the rabbits. This was evident from messy rabbit cages as feces were dispersed over the whole area of the cage. There was a definite lack of anal area hygiene as the area was always covered in a thin layer of feces. After implantation, the fecal hygiene returned to normal with a clean anal area and normal defecatory movement.

An improvement in defecatory activity was observed as early as 3 weeks after implantation of the bioengineered sphincters. Stool consistency returned to a firm pellet, similar to what was observed before FI was induced by the sphincterectomy.

#### *8.2.3.3 Histopathology assessment*

The post-implant harvested tissues displayed intact BioSphincter after 12 months of implantation. The presence of a thick continuous sheet of muscles innervated with neuronal network validated the manometry outcomes. There was the absence of any

*BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*

**Figure 2.**

differentiated functional neural network was confirmed via immune-reactivity against smoothelin and βIII tubulin. These results further validated via positive expression of smoothelin and βIII tubulin qPCR. Engineered IAS sphincters were tested for physiological functionality. The engineered tissues able to generate the spontaneous basal tone and exhibited a robust stable response following pharmacological or electrical stimuli. The bioengineered autologous BioSphincters were

Anorectal manometry is a technique used to measure contractility in the anus and rectum. Anorectal manometry was performed initially at baseline prior to any surgery. These measurements reflected the control state for all animals in this study. Anorectal manometry was performed prior to any surgery (before animals went for

sphincterectomy (biopsy), then at 3, 6, and 12 months in each experimental group.

IAS hemi-sphincterectomy resulted in a significant decrease in anal basal pressure and RAIR compared to baseline (no surgery), supporting the validity of the induced-incontinence model. In the sham surgery group, anal basal pressure and RAIR were not improved and were comparable to readings from rabbits in the nontreated group. Compared to baseline, the basal pressure in non-treated and sham group was decreased by 41% (p < 0.0001) after 1 month of hemi-sphincterectomy and remained low up to study time point of 12 months. Similarly, RAIR was also

non-treated group (49.2%) and sham groups (40.0%) compared to baseline till the

The IAS hemi-sphincterectomy affected fecal hygiene of the rabbits. This was evident from messy rabbit cages as feces were dispersed over the whole area of the cage. There was a definite lack of anal area hygiene as the area was always covered in a thin layer of feces. After implantation, the fecal hygiene returned to normal

An improvement in defecatory activity was observed as early as 3 weeks after implantation of the bioengineered sphincters. Stool consistency returned to a firm pellet, similar to what was observed before FI was induced by the sphincterectomy.

The post-implant harvested tissues displayed intact BioSphincter after 12 months of implantation. The presence of a thick continuous sheet of muscles innervated with neuronal network validated the manometry outcomes. There was the absence of any

This reduced anorectal functionality was restored within 1-month postimplantation of autologous BioSphincters in the treated group. The resting pressure was returned to baseline after 4 weeks of implantation and remained similar up to 12 months. RAIR was restored by 88% in initial 1 month and improved within 3 months and sustained till 12 months. The restoration of basal pressure and RAIR were significantly higher (p < 0.0001) than values observed in the non-treated

implanted adjacent to IAS tissues into the respective rabbits [10, 39].

any procedure) to record the baseline, and 1 month following IAS hemi-

reduced by 50.9% from the baseline (p < 0.0001). It remained low in

*8.2.3 Implantation and restoration of fecal continence*

*8.2.3.1 Restoration of anorectal pressure*

*Current Topics in Faecal Incontinence*

study time point.

group and sham groups.

*8.2.3.2 Improvement in fecal hygiene*

*8.2.3.3 Histopathology assessment*

**112**

with a clean anal area and normal defecatory movement.

*Different stages from bioengineered sphincter to implantation; (A) bioengineered sphincter; (B) implantation of two bioengineered sphincters; (C) 4 implanted bioengineered sphincters; and (D) implanted bioengineered sphincter after euthanasia (after 12 months of implantation).*

fibrosis or avascular collagen around the implant, indicating no foreign-body reaction with the implants. Pathologic findings in this study were generally minor and consisted primarily of a low incidence of background changes and minor changes attributable to implantation. There was no evidence of neoplasia. These results confirmed that the bioengineered sphincters were viable and functional in vivo with the maintenance of both the muscle and neural components [10, 39].

In this study, passive Fi was successfully developed in the large animal model. The bioengineered intrinsically innervated IAS constructs from the autologous cells retrieved at biopsy. The IAS constructs were bioengineered and implanted after 6– 8 weeks after harvesting the cells (**Figure 2A**); then, one by one, four bioengineered sphincters were implanted at the anal site (**Figure 2B**). The four bioengineered sphincters were stacked together at the site (**Figure 2C**). After 12 months of implantation, implanted bioengineered sphincters appeared intact as one tissue at the site (**Figure 2D**).

The animals resumed normal activity and defecatory bowel movement. There was no indication of any rectal outlet obstruction or anal stenosis. Anorectal manometry was performed on the animals monthly beginning 6 weeks after implantation. The animals exhibited a reinstated basal tone and RAIR. Animals were maintained and monitored up to 12 months after implantation. At each endpoint, after euthanasia, the harvested implant was tested. Results show that the construct maintained physiological functionality. The tests show that both muscle and neural type of cells maintained their physiological function. In other experiments, we have demonstrated that the cells of the implant stayed within the implant and did not migrate outside the location of the implant.

#### **9. Conclusion**

Regeneration of an intrinsically innervated function IAS sphincter is a promising approach for long-term relief from passive FI. The IAS muscle and neural cells synergized in collagen-laminin hydrogel as a 3D sphincter like architecture, mimicking the native IAS cell orientation and innervation. The bioengineering process has been optimized, scaled up for clinical application using human origin cells. The signaling pathways for sphincter tone and contraction were characterized. The bioengineered sphincter able to generate spontaneous tone and response to different pharmacological agents was comparable to human IAS. The stability, viability and cytocompatibility analysis of engineered sphincters were carried out in vitro and in vivo conditions. The step-wise pre-clinical assessment of engineered

autologous BioSphincters confirmed biocompatibility as IAS sphincter substitute, without any adverse effect. The implanted autologous BioSphincters vascularized, integrated with the impaired native IAS and regenerated stable, circularly oriented IAS muscle population, innervated with the neural network. The regeneration approach provided immediate symptomatic relief by restoration fecal hygiene. We have developed a large animal model of passive fecal incontinence and demonstrated sustained restoration of fecal continence, and restoration of basal tone and restoration of RAIR in this model after implantation of engineered autologous intrinsically innervated internal anal sphincter (IAS) BioSphincters. In a clinical scenario, this innovative approach will be able to reinstate continence, by providing an additive functional intrinsically innervated IAS bioengineered from the patient's cells.

**References**

[1] Rao SS. Pathophysiology of adult fecal incontinence. Gastroenterology. 2004;**126**(1 Suppl 1):S14-S22. DOI: 10.1053/j.gastro.2003.10.013

*DOI: http://dx.doi.org/10.5772/intechopen.86345*

*BioSphincter a Regenerative Medicine Approach to Treat FI*

[9] Bharucha AEA, Dunivan G, Goode PPS, et al. Epidemiology, pathophysiology, and classification of fecal incontinence: State of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) workshop. The American Journal of Gastroenterology. 2015;**110**(1):127-136. DOI: 10.1038/

[10] Bohl JL, Zakhem E, Bitar KN, Autonomic I, Neurological N. Successful treatment of passive Fecal incontinence in an animal model using engineered Biosphincters: A 3 month follow-up study. Stem Cells Translational Medicine. 2017;**6**(9):1795-1802. DOI:

[11] Huebner M, Margulies RU, Fenner DE, et al. Age effects on internal anal sphincter thickness and diameter in nulliparous females. Diseases of the Colon and Rectum. 2007;**50**(9):1405-1411. DOI:

10.1002/sctm.16-0458

10.1007/s10350-006-0877-7

2004;**47**(10):1643-1649

DOI: 10.1038/ajg.2014.303

[14] Bliss DZ, Jung HJ, Savik K, et al. Supplementation with dietary fiber improves fecal incontinence. Nursing Research. 2001;**50**(4):203-213. DOI: 10.1097/00006199-200107000-00004

[15] Omar MI, Alexander CE. Drug treatment for faecal incontinence in

[12] Lindsey I, Jones OM, Smilgin-Humphreys MM, Cunningham C, Mortensen NJ. Patterns of fecal incontinence after anal surgery. Diseases of the Colon and Rectum.

[13] Whitehead WE, Rao SSC, Lowry A, et al. Treatment of fecal incontinence: State of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases workshop. The American Journal of Gastroenterology. 2015;**110**(1):138-146.

ajg.2014.396

[2] SSCC R, Bharucha AE, Chiarioni G,

[3] Bartlett L, Nowak M, Ho Y-H. Impact of fecal incontinence on quality of life. World Journal of Gastroenterology. 2009;**15**(26):

Investigating and treating fecal

[4] Lazarescu A, Turnbull GK, Vanner S.

incontinence: When and how. Canadian Journal of Gastroenterology. 2009; **23**(4):301-308. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/

[5] Goode PS, Burgio KL, Halli AD, et al. Prevalence and correlates of fecal incontinence in community-dwelling older adults. Journal of the American Geriatrics Society. 2005;**53**(4):

[6] Engel AF, Kamm MA, Bartram CI, Nicholls RJ. Relationship of symptoms in faecal incontinence to specific sphincter abnormalities. International Journal of Colorectal Disease. 1995; **10**(3):152-155. DOI: 10.1007/

[7] Bharucha AE. Pelvic floor: Anatomy and function. Neurogastroenterology and Motility. 2006;**18**(7):507-519. DOI: 10.1111/j.1365-2982.2006.00803.x

[8] Rattan S. The internal anal sphincter: Regulation of smooth muscle tone and relaxation. Neurogastroenterology and Motility. 2005;**17**(Suppl 1):50-59. DOI: 10.1111/j.1365-2982.2005.00659.x

et al. Anorectal disorders. Gastroenterology. 2018;**150**(6): 1430-1442.e4. DOI: 10.1053/j.

gastro.2016.02.009

3276-3282

PMC2711681/

BF00298538

**115**

629-635. DOI: 10.1111/ j.1532-5415.2005.53211.x

As summary, regeneration, and implantation of the IAS BioSphincter will benefit a large socially distressed segment of the population via restoration of physiological function of the IAS, resolve FI, and improving quality of life.

#### **Acknowledgements**

This work was supported by NIH/NIDDK STTR R42DK105593.

### **Conflict of interest**

KNB is the founder of CELLF BIO LLC a startup biotech that has an interest in developing treatments for neurodegenerative diseases of the gut.

#### **Author details**

Prabhash Dadhich1,2 and Khalil N. Bitar1,2,3,4\*

1 Wake Forest School of Medicine, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA

2 Program in Neuro-Gastroenterology and Motility, Wake Forest School of Medicine, Winston Salem, NC, USA

3 Section on Gastroenterology, Wake Forest School of Medicine, Winston Salem, NC, USA

4 Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest School of Medicine, Winston Salem, NC, USA

\*Address all correspondence to: kbitar@wakehealth.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*BioSphincter a Regenerative Medicine Approach to Treat FI DOI: http://dx.doi.org/10.5772/intechopen.86345*

#### **References**

autologous BioSphincters confirmed biocompatibility as IAS sphincter substitute, without any adverse effect. The implanted autologous BioSphincters vascularized, integrated with the impaired native IAS and regenerated stable, circularly oriented IAS muscle population, innervated with the neural network. The regeneration approach provided immediate symptomatic relief by restoration fecal hygiene. We have developed a large animal model of passive fecal incontinence and demonstrated sustained restoration of fecal continence, and restoration of basal tone and restoration of RAIR in this model after implantation of engineered autologous intrinsically innervated internal anal sphincter (IAS) BioSphincters. In a clinical scenario, this innovative approach will be able to reinstate continence, by providing an additive functional intrinsically innervated IAS bioengineered from the patient's

As summary, regeneration, and implantation of the IAS BioSphincter will benefit a large socially distressed segment of the population via restoration of physio-

KNB is the founder of CELLF BIO LLC a startup biotech that has an interest in

logical function of the IAS, resolve FI, and improving quality of life.

This work was supported by NIH/NIDDK STTR R42DK105593.

developing treatments for neurodegenerative diseases of the gut.

1 Wake Forest School of Medicine, Wake Forest Institute for Regenerative

2 Program in Neuro-Gastroenterology and Motility, Wake Forest School of

3 Section on Gastroenterology, Wake Forest School of Medicine, Winston Salem,

4 Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Prabhash Dadhich1,2 and Khalil N. Bitar1,2,3,4\*

Forest School of Medicine, Winston Salem, NC, USA

\*Address all correspondence to: kbitar@wakehealth.edu

Medicine, Winston Salem, NC, USA

Medicine, Winston Salem, NC, USA

provided the original work is properly cited.

cells.

**Acknowledgements**

*Current Topics in Faecal Incontinence*

**Conflict of interest**

**Author details**

NC, USA

**114**

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[2] SSCC R, Bharucha AE, Chiarioni G, et al. Anorectal disorders. Gastroenterology. 2018;**150**(6): 1430-1442.e4. DOI: 10.1053/j. gastro.2016.02.009

[3] Bartlett L, Nowak M, Ho Y-H. Impact of fecal incontinence on quality of life. World Journal of Gastroenterology. 2009;**15**(26): 3276-3282

[4] Lazarescu A, Turnbull GK, Vanner S. Investigating and treating fecal incontinence: When and how. Canadian Journal of Gastroenterology. 2009; **23**(4):301-308. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/ PMC2711681/

[5] Goode PS, Burgio KL, Halli AD, et al. Prevalence and correlates of fecal incontinence in community-dwelling older adults. Journal of the American Geriatrics Society. 2005;**53**(4): 629-635. DOI: 10.1111/ j.1532-5415.2005.53211.x

[6] Engel AF, Kamm MA, Bartram CI, Nicholls RJ. Relationship of symptoms in faecal incontinence to specific sphincter abnormalities. International Journal of Colorectal Disease. 1995; **10**(3):152-155. DOI: 10.1007/ BF00298538

[7] Bharucha AE. Pelvic floor: Anatomy and function. Neurogastroenterology and Motility. 2006;**18**(7):507-519. DOI: 10.1111/j.1365-2982.2006.00803.x

[8] Rattan S. The internal anal sphincter: Regulation of smooth muscle tone and relaxation. Neurogastroenterology and Motility. 2005;**17**(Suppl 1):50-59. DOI: 10.1111/j.1365-2982.2005.00659.x

[9] Bharucha AEA, Dunivan G, Goode PPS, et al. Epidemiology, pathophysiology, and classification of fecal incontinence: State of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) workshop. The American Journal of Gastroenterology. 2015;**110**(1):127-136. DOI: 10.1038/ ajg.2014.396

[10] Bohl JL, Zakhem E, Bitar KN, Autonomic I, Neurological N. Successful treatment of passive Fecal incontinence in an animal model using engineered Biosphincters: A 3 month follow-up study. Stem Cells Translational Medicine. 2017;**6**(9):1795-1802. DOI: 10.1002/sctm.16-0458

[11] Huebner M, Margulies RU, Fenner DE, et al. Age effects on internal anal sphincter thickness and diameter in nulliparous females. Diseases of the Colon and Rectum. 2007;**50**(9):1405-1411. DOI: 10.1007/s10350-006-0877-7

[12] Lindsey I, Jones OM, Smilgin-Humphreys MM, Cunningham C, Mortensen NJ. Patterns of fecal incontinence after anal surgery. Diseases of the Colon and Rectum. 2004;**47**(10):1643-1649

[13] Whitehead WE, Rao SSC, Lowry A, et al. Treatment of fecal incontinence: State of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases workshop. The American Journal of Gastroenterology. 2015;**110**(1):138-146. DOI: 10.1038/ajg.2014.303

[14] Bliss DZ, Jung HJ, Savik K, et al. Supplementation with dietary fiber improves fecal incontinence. Nursing Research. 2001;**50**(4):203-213. DOI: 10.1097/00006199-200107000-00004

[15] Omar MI, Alexander CE. Drug treatment for faecal incontinence in adults. Cochrane Database of Systematic Reviews. 2013;(6):1-61. DOI: 10.1002/ 14651858.CD002116.pub2

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[17] Norton C, Chelvanayagam S, Wilson-Barnett J, et al. Randomized controlled trial of biofeedback for fecal incontinence. Gastroenterology. 2003; **125**(5):1320-1329. DOI: 10.1053/ S0016-5085(03)01368-4

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## *Edited by John Camilleri-Brennan*

Faecal incontinence is a highly prevalent condition that continues to have an impact on the activities of daily living of both men and women worldwide. This book, Current Topics in Faecal Incontinence, provides a comprehensive and up-to-date overview of some specialist areas in the diagnosis and management of this condition. The topics that are discussed include the evaluation of faecal incontinence, quality of life, faecal incontinence in autoimmune disease and in obstructive defaecation, anal implants, and biosphincters. This book is an invaluable resource for physicians, surgeons, nurses, and allied healthcare professionals who seek to expand and refresh their knowledge in this field, as well as a source of excellent information for those preparing for professional examinations.

Published in London, UK © 2020 IntechOpen © Model-la / iStock

Current Topics in Faecal Incontinence

Current Topics in

Faecal Incontinence

*Edited by John Camilleri-Brennan*