Section 3 Bioengineering

**43**

**Chapter 3**

Vehicles

*James Melrose*

**Abstract**

Novel Approaches in Meniscal

Repair Utilizing Mesenchymal

Mesenchymal stem cells (MSCs) have been widely applied in the repair of the knee-joint menisci which have a limited ability to undergo spontaneous repair. The menisci stabilise the knee-joint and are weight-bearing structures subjected to considerable tensional and compressive forces during flexion-extension and torsional loading of the knee. Traumatic loading of the knee-joint menisci can generate a number of lesions in the inner avascular meniscal regions. These have a limited capability of intrinsic repair and predispose the underlying articular cartilages to premature osteoarthritis. A number of strategies have therefore been developed for meniscal repair employing MSCs, bioscaffolds, hydrogels, biological glue cell delivery systems and agents which promote cell proliferation/matrix synthesis. Meniscal implants have also been developed in combination with the above procedures. It is important that meniscal defects be repaired not only to maintain kneejoint stability but also to prevent further degenerative changes in other knee joint tissues. Degenerative menisci contribute degradative proteinases and inflammatory mediators to the total synovial degradative proteinase pool. Partial or total surgical removal of the menisci is not a solution since this leads to premature osteoarthritis. Meniscal integrity needs to be maintained or repair strategies implemented in a

**Keywords:** meniscal repair, mesenchymal stem cell, bioscaffolds, biological glues,

The knee joint menisci provide joint stability during weight bearing, the curved superior meniscal surfaces provide congruity between the curved femoral condyle and flat tibial articular cartilages [1]. The menisci act as shock absorbers and protect

Stem Cells, New Generation

Bioscaffolds and Biological

Adhesives as Cell Delivery

timely manner to maintain knee joint function.

meniscal implants/allografts

**1.1 Meniscal structure: function**

**1. Introduction**

#### **Chapter 3**

 Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation Bioscaffolds and Biological Adhesives as Cell Delivery Vehicles

*James Melrose*

### **Abstract**

Mesenchymal stem cells (MSCs) have been widely applied in the repair of the knee-joint menisci which have a limited ability to undergo spontaneous repair. The menisci stabilise the knee-joint and are weight-bearing structures subjected to considerable tensional and compressive forces during flexion-extension and torsional loading of the knee. Traumatic loading of the knee-joint menisci can generate a number of lesions in the inner avascular meniscal regions. These have a limited capability of intrinsic repair and predispose the underlying articular cartilages to premature osteoarthritis. A number of strategies have therefore been developed for meniscal repair employing MSCs, bioscaffolds, hydrogels, biological glue cell delivery systems and agents which promote cell proliferation/matrix synthesis. Meniscal implants have also been developed in combination with the above procedures. It is important that meniscal defects be repaired not only to maintain kneejoint stability but also to prevent further degenerative changes in other knee joint tissues. Degenerative menisci contribute degradative proteinases and inflammatory mediators to the total synovial degradative proteinase pool. Partial or total surgical removal of the menisci is not a solution since this leads to premature osteoarthritis. Meniscal integrity needs to be maintained or repair strategies implemented in a timely manner to maintain knee joint function.

**Keywords:** meniscal repair, mesenchymal stem cell, bioscaffolds, biological glues, meniscal implants/allografts

#### **1. Introduction**

#### **1.1 Meniscal structure: function**

 The knee joint menisci provide joint stability during weight bearing, the curved superior meniscal surfaces provide congruity between the curved femoral condyle and flat tibial articular cartilages [1]. The menisci act as shock absorbers and protect the weight bearing articular tissues from excessive point loading [2] transferring forces between the femoral and tibial joint surfaces, transmitting 50–90% of the total knee joint load during weight-bearing [3, 4]. The structural organisation of the meniscus is designed to withstand circumferential hoop stresses which are generated within the meniscal tissue to dissipate tensile stresses which are transferred along the circumferential meniscal collagen fibre networks counteracting the tendency of the menisci to be extruded peripherally when the knee joint is subjected to compressive loading [5]. Energy is absorbed into the collagen fibres by the dynamic expulsion of joint fluid from the aggrecan-hyaluronan macro-aggregate networks entrapped within the meniscal collagenous networks. The menisci are fibre reinforced structures stiffening and protecting them from damage by excessive deformation during compressive loading [6] (**Figure 1a** and **b**).

The contribution of intact menisci in knee load-bearing is emphasised from the increase in contact forces in the underlying articular cartilages of up to 350% following partial or total meniscectomy where as little as 16–34% of the intact meniscus may be removed [1, 3, 7]. Radial meniscal tears which extend to its periphery may result in significant contact forces being transmitted to the underlying articular cartilages which can damage these tissues [8].

 Water (~70% wet weight) and collagen, (mainly type I, and lower amounts of type II, III and VI collagen constitute 60–70% of the meniscal dry weight) are major meniscal components [9–15]. Proteoglycans (aggrecan, decorin, biglycan, versican, fibromodulin, lumican, keratocan) and elastic microfibrillar glycoproteins are quantitatively minor meniscal extracellular matrix (ECM) components but convey essential functional properties [14–16]. The meniscus is a complex fibre-reinforced structure designed to withstand multidirectional tensional and compressive forces (**Figure 1a** and **b**). The outer third of the meniscus (red zone) is served by a fine capillary network. Defects in this region of the meniscus have the ability to undergo spontaneous repair however the inner two thirds of the meniscus (white zone) is avascular and has a limited ability to undergo repair (**Figure 2a**). The outer zone of the meniscus is a collagen rich fibrocartilaginous tissue while the inner zone contains higher proteoglycan levels and is cartilaginous (**Figure 2b**). Immunolocalisation of perlecan, HSPG2, a large modular HS multifunctional proteoglycan demonstrates a strong localisation pattern in this inner region. Perlecan is marker of chondrogenesis [17–20].

Supraphysiological overload of the menisci may generate defects in the inner meniscus diminishing its weight bearing capability and ability to resist tensional stresses and it becomes less able to dissipate such forces to prevent overloading of the underlying articular cartilage. A number of characteristic tears (buckethandle, degenerate) occur in the inner meniscal region. Longitudinal and radial tears can also affect the outer meniscus (**Figure 2c**). This can also damage the underlying articular cartilages formerly protected by the menisci leading to degenerative changes and impacting on the knee's ability to act efficiently as an articulating weight bearing structure. Development of premature osteoarthritis (OA) may also result in such circumstances [21, 22]. Menisci in OA knees are also subject to ectopic focal depositions of calcium in cyst like structures (**Figure 2d**). Fibrillation of the inner meniscal region is also a common degenerative feature in OA. Meniscal cell clustering adjacent to such fibrillations is also common and may indicate endogenous adult stem cell activity in response to altered biomechanics/nutrition in this region. Cell clustering has also been observed adjacent to surface fibrillations in OA articular cartilage and adjacent to lesions in the annulus fibrosus of the degenerate intervertebral disc [23–29]. Such cell clustering may be indicative of an incomplete frustrated repair response by resident adult stem cells.

*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 

#### **Figure 1.**

*Diagrammatic representation of the collagenous organisation in a meniscus. (i) The meniscus contains a complex arrangement of radial collagen fibre bundles in the outer meniscus, (ii) thick radial tie bundles internally as well as (iii) finer collagen fibre bundles of collagen in lamellar sheets in the inferior and superior meniscal surfaces. Notice that the inferior lamina is significantly thicker than the superior lamina. Vertical radial sections through 2 year old lateral and medial ovine menisci stained with picrosirius red and viewed under polarised light depicting collagen bundles which are highly refractile due to their ordered collagenous structure appearing as bright rod-like structures (b). Picrosirius red predominantly visualises the major fibrillar meniscal collagen, type I collagen. Methodology for Picrosirius red staining is as described earlier [78].* 

Many strategies have consequently been developed to effect meniscal repair using a number of cell types including mesenchymal stem cells (MSCs) sourced from a number of tissues (**Table 1**), and combinations of bioscaffolds, hydrogels,

#### **Figure 2.**

*Structural features evident in the normal and degenerate meniscus. Diagrammatic representation of the vascularisation of a vertically sectioned meniscus showing the extensive capillary network in the outer meniscal red zone and lack of a blood supply to the inner two thirds of the meniscus (a). The inner meniscus is a cartilage like tissue which is well delineated in a newborn meniscus by immunolocalisation of perlecan, HSPG2, a chondrogenic marker proteoglycan (b). Menisci are subject to a number of structural defects which are summarised diagrammatically (c). Histochemical visualisation (H & E) and toluidine blue staining, of some features of degenerate menisci (d). Focal deposition of small calcium deposits in a cyst like formation in the outer meniscus zone in a 53 year old human meniscus. Fibrillation of the inner meniscal zone and cell cluster formation. In the normal meniscus single cells are distributed throughout the meniscus with no clustering.* 

bioadhesive cell delivery systems and bioactive agents which stimulate the resident and exogenous cells applied for therapeutic purposes (**Tables 2** and **3**).

 In-vitro experiments have shown that co-culture of bone marrow derived stromal stem cells with meniscal cells increases cell proliferation and matrix synthesis [30]. Type I and type II collagen and aggrecan mRNA expression were elevated and ECM protein levels increased (**Figure 3a** and **b**). Significantly, meniscal cells stimulated with FGF-2 or FGF-18 in 3D pellet culture also produced elevated levels of these ECM components (**Figure 3c** and **d**). MSCs are believed to act both through transfer of material directly to resident cell populations through cell-cell contact

*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 


#### **Table 1.**

*Mesenchymal stem cell (MSC) sources used in therapeutic approaches for meniscal repair.* 


#### **Table 2.**

*Meniscal allografts and implants used for meniscal repair and replacement.* 



*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 


*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 


*Abbreviations: PRP, platelet rich plasma; rAAV, recombinant Adeno-Associated Virus; PGA, polyglycolic acid; HYADD4®, hyaluronan derivative; PLGA, polylactic-co-glycolic acid, CTGF, connective tissue growth factor; VEGF, vascular endothelial cell growth factor; OP-1, osteogenic protein-1; CS, chondroitin sulphate; FGF-2, fibroblast growth factor-2; TGF-β, transforming growth factor-β.* 

#### **Table 3.**

*Meniscal repair using bio scaffolds, bioactive substances and bio adhesives.* 

and also by secretion of trophic factors which both stimulate tissue regenerative processes [31–42]. Co-cultures of synovial stem cells [43] and MSCs [44–48] with meniscal cells have been evaluated in a number of biomatrices for meniscal repair purposes (**Table 1**).

#### **Figure 3.**

*Co-culture of meniscal cells and bone marrow derived mesenchymal stromal cells induces cell proliferation and ECM production and is recapitulated to some degree by treatment of meniscal cells with FGF-2 and FGF-18. Immunolocalisation of meniscal matrix components in micro-mass pellet culture. Immunolocalisation of type I collagen, type II collagen and aggrecan (ACAN) in meniscal-MSC micro-mass pellet co-cultures (a). Negative controls of pellets using rabbit IgG (MSC pellet) and mouse IgG (meniscal cell pellet) for immunolocalisation in the absence of primary antibody. Anti-type I collagen (clone I-8H5) and anti-type II collagen (clone II-4CII) were from MP Biomedicals, Ohio, USA. A rabbit polyclonal antibody (pAb) # 2194 to aggrecan G1 domain was a gift from Dr. J Mort Joint Diseases laboratory, Shriners, Hospital for Children, McGill University, Montreal, QC, Canada [218]. PAb 2194 was raised against a mixture of four aggrecan specific G1 peptide-ovalbumin conjugates including HDNSLSVSIPQPSGGC, RVLLGTSLTIPCYFIDPMHPVTTAPS, TEGRVRVNSAYQDKGGC and SSRYDAICYTG (single letter amino acid code). Morphometric image analysis of meniscal matrix components produced in pellet culture. Quantitation of type I and type II collagen and aggrecan immunolocalisation levels in meniscal: MSC co-cultures using Adobe Photoshop CS4 morphometric image analysis software as integrated pixel density. Mean values ± SD for 3 pellet sections is shown (b). Immunolocalisation of matrix components produced by meniscal cells in pellet cultures stimulated with FGF-2 and FGF-18. Immunolocalisation of Type I and Type II collagen and aggrecan (ACAN) in meniscal cell micromass pellet cultures stimulated with FGF-2 and FGF-18 (a–c) for 21 days (c). Morphometric image analysis of meniscal matrix components using Adobe Photoshop CS4 morphometric image analysis software (d).* 

#### **2. Meniscus preserving therapies**

#### **2.1 Why it is important to preserve the knee joint meniscus? A historical perspective**

The meniscus was historically considered a vestigial muscle remnant and little importance was attributed to this structure for knee joint function. Consequently, radical surgery and total removal of the meniscus were common surgical practice in the 1980s with serious long-term consequences for the meniscectomised knee. It should have been obvious from meniscectomy studies used to induce OA

#### *Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552*

 experimentally in animals that surgical removal of the menisci from knee joints was not a benign procedure [49–74]. However it took time for these animal findings to be translated to human studies [59–61, 65, 67, 70, 72] and for these experimental findings to be fed through to human clinical practice and the importance of the meniscus in entirety in knee joint articulation, weight bearing and load distribution became established. Even so, publications were still appearing as late as 2016 emphasising the importance of the preservation of the knee joint menisci to ensure optimal knee joint function three decades after meniscal removal had been shown to induce degenerative changes in other knee joint tissues [75].

Currently, the consensus in the surgical treatment of meniscal tears is to preserve as much functional meniscal tissue as possible to preserve knee joint function [76].

The menisci play critical protective roles for the knee joint articular cartilages through shock absorption and load distribution and also have important roles to play in proprioception and balance [5]. The ESSKA (European Society for Sports Traumatology, Knee Surgery and Arthroscopy) MENISCUS CONSENSUS INITIATIVE was initiated in 2014 to find a European consensus on the treatment of meniscus pathologies [76].

 Further studies in animals [73, 77–79] established a more direct contribution from meniscal degeneration to joint structures globally during degenerative conditions such as OA and RA. During the development of arthritic conditions in animals [73, 77, 79] and humans [80] tissue proteoglycans become fragmented through proteolytic degradation and this reduces the weight bearing and articulatory properties of the articular cartilages and menisci and may even impact on subchondral bone [80]. Matrix metalloproteases (MMPs), ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs)-4 and ADAMTS-5 produced by articular chondrocytes have a major impact on aggrecan and other cartilage proteoglycans reducing the weight bearing properties of the knee joint articular cartilages. The increase in synovial degradative protease pool during OA and RA was previously attributed to the articular chondrocytes which respond to inflammatory cytokines in the arthritic joint by producing these degradative proteases. Recent in-vitro studies have however now shown that meniscal fibrochondrocytes also potently respond to interleukin-1 and tumour necrosis factor-α by producing significant levels of MMPs (MMP-1, 2, 3, 9, 13), ADAMTS-4 and ADAMTS-5 and are a major cellular source of these components in the total global degradative enzyme pool present in synovial fluid [81–83]. Meniscal cells actually produce higher levels of these degradative components than articular chondrocytes, thus represent a previously unidentified therapeutic target in the treatment of OA and RA.

#### **2.2 Meniscal implants**

 Partial or total meniscal replacement by collagen or synthetic allografts following meniscectomy have yielded mixed results (**Table 2**) [84, 85]. Implants fall into two categories, (i) porous, resorbable implants which stimulate tissue regeneration and (ii) solid, non-resorbable implants which physically replace the meniscus [86]. The Regen Menaflex™ collagen total meniscal implant (CMI®, Ivy Sports Medicine) is a resorbable implant. A review of the CMI® by Hansen et al. in a 10 year follow up confirmed good clinical outcomes, solid integration of the CMI® with host tissue and it was concluded that the CMI® held promise for meniscal repair [87]. After a protracted series of re-reviews of experimental data, technical issues and protocols the FDA rescinded approval for the Menaflex® device in 2013. The Actifit® polymeric polyurethane partial implant (ORTEQ Sports Medicine) is a honeycomb scaffold that enables blood-flow through it providing a route for cellular in-growth as the body's natural healing process takes place. Once the damaged section of the

meniscus surgically removed the implant is attached to an area of the remaining meniscus with a good blood supply [86]. This has improved knee joint function and reduced knee pain in patients for up to 5 years after implantation and a stable cartilage profile was achieved in 46.7% of patients but a relatively high failure rate was also reported [88–90].

An artificial Polycarbonate-urethane implant has been developed for replacement of the medial meniscus [91–93]. NUsurface® have developed a polyethylene reinforced polycarbonate urethane total meniscal implant, approved for use in Europe since 2008 and in Israel since 2011 [94]. The safety and long-term performance of the NUsurface implant is currently under evaluation in SUN (Safety Using NUsurface®) and VENUS (Verifying the Effectiveness of the NUsurface® System) clinical trials in the USA.

Salt modified cross-linked PVA based hydrogels seeded with meniscal cells have been evaluated for meniscal repair [95] as have polyglycolic acid implants seeded with chondrocytes [96] and (poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) meniscal implants seeded with fibrochondrocytes [97].

 Biodegradable thermoplastic polyurethane Estane® polymer (Lubrizol Corp, USA) porous implants have been evaluated in dogs as a meniscal replacement [98]. Colonisation of the implant by resident meniscal synovial cells from the peripheral attachments, laying down of matrix components within the implant and the biointegration of the implant to the peripheral meniscal attachment tissues were evaluated 3–6 month post implantation. This demonstrated that the implant filled completely with meniscal tissue as demonstrated by toluidine blue staining for proteoglycan, and for type II collagen and I by immunolocalisations using specific collagen antibodies. Histological evaluation of the tibia and femoral articular cartilages confirmed these tissues did not degenerate in the experimental period employed for this study.

 A number of critical reviews on the performance of meniscal implants [86, 87, 99–101] generally acknowledge that despite initial promising findings long-term and randomised controlled studies still need to be undertaken to confirm implant performance and reliability for meniscal repair and that the development of a meniscal replacement tissue of comparable performance to native tissue has yet to be achieved.

#### **2.3 3D printing of knee joint menisci**

Polycaprolactone has been used as a scaffolding material to form an exact meniscal replica using a 3D printer [102–105]. MRI scans of the meniscus are converted into a 3D image, data from this image is then used to drive a 3D printer, which produces a scaffold in the exact shape of the meniscus, down to a resolution of 10 μm. Differential release of CTGF and TGF-β3 to drive formation initially of the outer collagenous meniscal region then the more cartilaginous inner meniscus is achieved by slow release microspheres containing CTGF and TGF-β3 in the printed meniscus. These attract meniscal progenitor cells into the scaffold which lay down tissue gradients to form the collagenous outer and cartilaginous inner regions of the meniscus. In sheep this takes between 4 and 6 weeks to achieve meniscal replacement and the scaffolding material then slowly redissolves to be eliminated by normal resorptive processes.

Interpenetrating networks of poly(2-acrylamido-2-methylpropanesulfonate) and polyacrylamide can be prepared by varying the ratio of polyacrylamide to cross-linker, to yield a gel with compression strength and elastic modulus of 61.9 and 0.44 MPa. This gel has maximum compressive and tensile strengths of 93.5 and 1.4 MPa respectively. This can be used in a 3D printer to prepare replacement *Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 

menisci from a patients X-ray computed tomography image of a meniscus [104]. Slow release of CTGF and TGF-β3 from a 3D printed meniscus stimulated endogenous stem/progenitor cells to undertake meniscal regeneration [106].

#### **3. Meniscus regenerative therapies**

#### **3.1 Therapeutic use of mesenchymal stem cells in tissue repair**

Mesenchymal stem cells (MSCs) have been the subject of intense investigation since their discovery in the 1960s due to their remarkable efficacy in tissue repair. MSCs were originally considered to migrate into sites of injury, where they engrafted, and differentiated into functional cells, resulting in regeneration of damaged or diseased connective tissue [107]. Findings from several hundred animal studies and many human clinical trials have challenged this mode of action. MSCs certainly exhibit a remarkable ability to repair diseased tissues, but it has become increasingly apparent that they do not engraft in enough numbers or for sufficient durations in tissue defects to provide tissue repair and clinical benefit directly. Additional modes of action for MSCs have therefore been proposed based on their ability to enhance resident cell viability and/or proliferation, reduce cell apoptosis [108, 109], and, in some cases, modulate immune responses [110–114]. These are due to paracrine effects due to secreted growth factors, cytokines, and hormones by the MSCs and cell-cell interactions mediated through communicating nanotubes, which convey extracellular vesicles containing reparative peptides/proteins, mRNA, and microRNAs [107]. Caplan (2017) has proposed that stem cells should be renamed *Medicinal Signalling Cells* to more accurately reflect how they home in on injured or diseased tissue sites secreting bioactive factors with immunomodulatory and trophic properties which direct the resident cells to undertake the tissue repair process, this may happen long after the MSCs have disappeared from the defect site [115].

MSCs have gained popularity for tissue repair with good reason [32, 116], and several applications have been developed for their use in the repair of connective tissues including the meniscus [117–125].

#### *3.1.1 How do MSCs effect tissue repair?*

Despite their widespread use in therapeutic applications the precise mode of action of MSCs remains elusive [126–130]. MSCs undergo engraftment in a defect site and differentiate to an appropriate cell lineage conducive to tissue repair [131] where they act as in-situ reservoirs of trophic factors [132] which direct resident cell populations to effect tissue repair [33, 40, 133–135]. It is un-resolved whether cell-cell contact is essential for MSC action in tissue repair [33, 117, 131]. The pluripotency of MSCs facilitates the differentiation of the engrafted cells to effect tissue repair [33, 133]. However, some evidence shows that only a small proportion of the MSCs actually integrate and survive in the host tissues and the predominant mechanism by which MSCs participate in tissue repair appears to reside in their paracrine activity through the production of a multitude of growth factors and cytokines [33, 132]. Lipid micro vesicles released by MSCs have also been shown to be an important means of cellular communication and occurs alongside the mediators secreted by the MSCs. Nano vesicles/exosomes transfer proteins, lipids and small RNAs to neighbouring cells, and through these mediate a variety of biological responses in addition to those mediated by soluble trophic factors supplied by the MSCs [35, 136, 137].

#### **3.2 Use of MSCs and chondrocytes for meniscal repair**

The use of meniscal, chondrocytes or MSCs [138] in tissue engineering [139] using synthetic and biological scaffolds [101] containing bioactive factors [140] hold promise in the repair of the meniscus. Direct intra-synovial injections of MSCs have also been employed and meniscal regeneration and resolution of pain recorded [135, 141]. MSCs sourced from a number of tissues including synovial tissues [142–145], adipose [146, 147], bone marrow [45, 148–150] and blood vessels [151] have been applied in a number of applications to promote meniscal repair [44–48, 152–158] (**Table 1**). Co-cultures of meniscal cells and MSCs have also been examined in meniscal repair strategies [43, 159, 160]. Furthermore, a diverse range of bio scaffolds have been developed containing CS have been developed to promote MSC differentiation in-vivo for varied applications in repair biology [161] (**Table 3**). These scaffolds are also appropriate for strategies aimed at meniscal repair but have yet to be applied in this area.

#### **3.3 Co-culture of MSCs/meniscal cells and in-vitro stimulation with FGF-2/FGF-18**

 MSCs hold tremendous promise in regenerative medicine however their mode of action remains to be precisely established. Direct cell-cell transfer of stem cell material to resident cells has been shown to promote tissue repair processes, while soluble trophic factors secreted by the stem cells can also stimulate repair. In order to examine these possibilities further in the meniscus, bone marrow MSCs and meniscal cells have been co-cultured in micro-mass pellet cultures (**Figure 3a**  and **b**). The influence of FGF-2 and FGF-18 on meniscal pellet cultures have also been assessed to mimic the action of soluble trophic factors (**Figure 3c** and **d**). Immunolocalisation of the extracellular matrix (ECM) components type I and II collagen and aggrecan (ACAN) have been used to assess the response of the meniscal cells to these treatments. Meniscal cell proliferation is significantly elevated by MSC co-culture, and deposition of type I collagen and type II collagen and ACAN elevated. FGF-2 and FGF-18 also increase these ECM components in pellet culture. Cross-talk between meniscal cells and MSCs (and FGF-2 and FGF-18 to a lesser extent) thus positively influence cell proliferation and matrix production conducive to tissue replenishment and repair which would be expected to be re-capitulated in-vivo upon administration of stem cells to meniscal defects. Thus direct cell-cell contact and soluble trophic factors both stimulate meniscal repair processes.

#### **3.4 Bioscaffolds, bioactive substances and bioadhesives and meniscal repair**

 The outer and inner meniscus have widely differing repair capability correlating with their relative blood supply [162, 163] (**Figure 1a**). The inner meniscus has the poorest blood supply and consequently the weakest repair response. Many strategies have focussed on the development of measures to improve repair of the inner meniscus and they fall into three broad categories: (i) mesenchymal stem cells administered by direct intra-articular injection; (ii) bioscaffold, hydrogel or bioadhesive cell delivery vehicles for the delivery of chondrocytes, meniscal cells or MSCs into meniscal defects; and (iii) meniscal implants and allografts for total or partial meniscal replacement. These procedures are often undertaken with bioactive substances in the scaffold, hydrogel or bioadhesive delivery system which stimulate repair processes in therapeutic and resident cell populations (**Table 3**). An alternative approach is the co-culture of MSCs with chondrocytes or meniscal cells to pre-condition these or expand cell numbers prior to their incorporation

#### *Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552*

into bioscaffolds, hydrogels or bioadhesives prior to administration to the meniscal defect [159, 164] (**Figure 3a** and **b**). Platelet rich plasma or platelet rich fibrin clots have been used to enhance meniscal repair in bioscaffolds [120, 165–171].

 Myoblast loaded PLGA scaffolds have been evaluated for the repair of inner meniscal defects [172]. A derivatised HA, HYADD4® hydrogel cell delivery system has been used for the repair of radial-longitudinal tears in a randomised controlled study [173]. Electrospun type I collagen and gelatin-PLGA bilayered nanofibre reinforced scaffolds seeded with meniscal cells isolated from outer and inner regions have been used in the repair of lesions in the inner meniscus [174, 175] and radioopaque collagen scaffolds have been used in order to observe the action of therapeutic cells including MSCs on meniscal repair [176]. Meniscal defects wrapped in collagen membranes prior to injection of autologous chondrocytes for repair have been evaluated for the repair of the avascular meniscus [156]. Collagen gel scaffolds containing meniscal, synovial and adipose stem cells have been employed for meniscal repair [177] or in electrospun nanofibrous scaffolds [178]. The use of a type I collagen scaffold and infrapatellar fat pad for meniscal repair has been evaluated in rabbits [179]. PLGA mesh and fibre reinforced collagen-GAG scaffolds seeded with chondrocytes [180] or meniscal cells [168] supplemented with PRP have been evaluated for meniscal repair. Minced juvenile menisci sandwiched with meniscal explants from inner meniscal regions have been evaluated for their reparative potential on tears of the inner meniscal regions [181]. A number of bioactive factors have been evaluated for their reparative properties on meniscal defects. These include multiple injections of leuko-reduced PRP [165], 10% human serum, 5% PRP, 5% autologous plasma [182]. Over expression of TGF-β induced by a rAAV vector, stimulated matrix production and cell proliferation in human meniscal explants consistent with active repair [183]. IGF-I over-expressing meniscal cells induced by transfection of the hIGF-I gene [184] or by liposome Fugene 6 transfer of hIGF-I, stimulated ECM production, proliferation and differentiation of cultured meniscal cells and explants from the inner meniscus [185]. VEGF, BMP-7 and HA stimulated chondrocytes have been implanted into meniscal defects to undertake repair in-vitro [186]. Intra-articular injection of microRNA 210 stimulated mitochondrial activity and angiogenesis promoting repair of avascular meniscal defects by upregulation of anabolic matrix genes by resident meniscal cells, VEGF and FGF-2 production [187]. Fibrin-CTGF administration into avascular defects stimulated repair by the resident meniscal cells [188] as did HA, TGF-β3, platelet concentrates and serum supplemented scaffolds [166, 182, 189]. FGF-2 over-expressing meniscal cells [190, 191] and gelatin-FGF-2 scaffolds [192] also stimulated repair of inner meniscal defects. HA-collagen-PRP composites [47, 193], VEGF [194], TGF-β1 [195] and OP-1 [196] also stimulated meniscal cells and MSCs to undertake repair of inner meniscal defects or punch biopsy wounds in menisci. The bioscaffolds used in meniscal repair or regenerative strategies have been extensively reviewed [197–200].

#### **3.5 Bioadhesives and meniscal repair**

First generation fibrin sealant/glue formulations (Tisseel® (Baxter International Inc.), Tissucol® (Baxter Healthcare SA), Beriplast® (CSL Behring GmbH), Hemaseel® (Haemacure Corp)) were originally based on bovine fibrinogen, thrombin and aprotinin isolated from pooled bovine donors. With the discovery of bovine spongiform encephalitis and the technical difficulty of removing prions from bovine protein products, second generation fibrin glues were developed using human proteins and in-house methodologies for the isolation of autologous platelet plasma. Vitagel® (Orthovita Inc.)/Costasis® (Angiotech Pharmaceuticals Inc.) is a fibrin sealant variant containing bovine collagen and thrombin and human

 plasma. To minimise transmission of viral components, second generation fibrin sealants/glues utilise heat-treated human fibrinogen, autologous platelet plasma and virally incapacitated human thrombin. Autologous fibrin sealants based on platelet rich plasma (PRP), or platelet poor plasma (PPP) with added calcium and thrombin, produce a platelet gel which promotes haemostasis and wound healing aided by the release of platelet growth factors (especially TGF-β1 and TGF-β2) and cytokines. Autologous fibrin sealants suffer inconsistency due to variation in patient plasma protein profiles. Commercial FDA approved second generation fibrin sealants such as Quixil® (OMRIX Biopharmaceuticals SA)/Crosseal™ (OMRIX Biopharmaceuticals) have controlled levels of fibrinogen and thrombin with aprotinin replaced by the anti-fibrinolytic, tranexamic acid. Concerns over the use of tranexamic acid subsequently led to it being dropped from the formulation in the product Evicel® (Ethicon HCP). Formulations of fibrin sealants/glues have been developed as aerosol administered foams and collagen films based on equine collagen and combinations of animal (Tachocomb® (Baxter Healthcare Corp)) and human fibrinogen/thrombin (Tachocomb H®, TachoSil® (Baxter Healthcare Corp)). While fibrin sealants/glues were originally developed to minimise surgical blood loss and to aid in wound repair they have now been applied as autologous cell delivery vehicles for osteochondral repair in autologous chondrocyte implantation (ACI) whereby chondrocyte numbers are expanded in-vitro then loaded into cartilage defects and are contained within this site using a periosteal or collagen membrane sutured over the defect site and sealed along its margins using fibrin sealants/glues. This technique was subsequently modified using the matrix assisted chondrocyte implantation (MACI) procedure where chondrocytes seeded into a matrix material were placed into the chondral defect and sealed in place with fibrin sealant/glue obviating the use of sutures. A modification of this procedure (fibrin ACI) where fibrin sealants were used as scaffolds for cell delivery has also been developed. The fibrin ACI methodology has been applied to the repair of meniscal tears [201–203] using a number of bioactive supplements to improve cell proliferation and matrix synthesis to promote meniscal repair.

An interesting novel bio-glue has been discovered in the Australian frog genus *Notaden bennetti*. During the mating season the female frog expresses an adhesive exudate from the dorsal skin which ensures sexual union with the male for an extended period to ensure effective fertilisation. This exudate has been harvested from frog skin by electro-stimulation and characterised. Examination of the toxicity and biocompatibility of this biological glue [204], its molecular composition and mechanism of action [205] has shown that this protein based adhesive [206] is non-immunogenic, biocompatible, displays elastomeric properties similar to elastin and the strength of its adhesive properties is several fold that of fibrin glue. This frog glue has been used in combination with suturing of infraspinatus tendon to the bone interface in rotator cuff operations and significantly increased the strength of these attachments [207]. The frog glue also outperformed fibrin glue for the re-attachment of the cut surfaces of a longitudinal bucket handle meniscal tear in an in-vitro comparison [208, 209]. Marine sources of biological glues from the New Zealand green lipped mussel and barnacle are known and have appropriate strong adhesive properties for orthopaedic applications, these await commercialisation [210–213].

CS-bone marrow tissue adhesive [214], fibrin stabilised PGA scaffolds [189] have both found application in meniscal repair. New generation bio-glues has been used as cell delivery vehicles and as bioadhesives in meniscal repair [210, 211] and in the re-attachment of horizontal meniscal defects [215]. Mussel based bioadhesives containing antibiotics and fungicides with improved wet strength properties for use in the closure of surgical incisions have even been developed [216, 217].

*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 

### **4. Conclusions**


#### **Acknowledgements**

NHMRC Project Grant 1004032 funded this study.

*Meniscus of the Knee - Function, Pathology and Management* 

#### **Author details**

James Melrose1,2,3,4\*

1 Raymond Purves Bone and Joint Research Laboratory, Kolling Institute, Northern Sydney Local Health District, St. Leonards, NSW, Australia

2 Sydney Medical School, Northern, The University of Sydney, Australia

3 Faculty of Medicine and Health, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW, Australia

4 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, Australia

\*Address all correspondence to: james.melrose@sydney.edu.au

© 2018 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.

*Novel Approaches in Meniscal Repair Utilizing Mesenchymal Stem Cells, New Generation… DOI: http://dx.doi.org/10.5772/intechopen.80552* 

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**77**

**Chapter 4**

**Abstract**

cus tissue replacement.

**1. Introduction**

Bio-Engineered Meniscus for

*Azran Azhim, Najian Ibrahim and Fatihah Yusof*

Meniscus plays fundamental roles in the knee mechanisms and functions. It acts as a shock absorber where it enables even distribution of forces, and also lubricates knee joints. Meniscal injuries could result to the onset of degenerative osteoarthritis if proper treatments are delayed. To date, treatment of meniscal injuries are more towards conservative methods and surgical approach commonly known as meniscectomy. Attempts to develop scaffolds for meniscus implants from synthetic and biological sources have been done in the recent years. This approach involves a multidisciplinary study known as tissue engineering and regenerative medicine. It involves the combination of three crucial aspects; the choice of chondrogenic/stem cells, bioscaffolds and favourable environmental factors such as growth factors. This chapter discusses and highlights on the currently available meniscal scaffolds that have been explored before. Focus is also directed on the potential of decellularized extracellular matrix (ECM), prepared through sonication treatment that produced scaffolds which mimics natural meniscus. The evaluation of decellularized scaffolds was portrayed through recellularization using cells namely chondrocytes, fibrochondrocytes and stem cells in order to regenerate new functional tissue. In short, this chapter serves as a representation of current approaches aiming in bio-engineering the meniscal scaffolds as menis-

**Keywords:** meniscus, bioscaffolds, decellularization, recellularization, implant

and injuries. There are three crucial aspects in determining the success of this approach which is by combining cells, biomaterial scaffolds and biologically active molecules such as growth factors. Developing three-dimensional scaffolds or constructs that could serve similarly as native tissue is utterly important. A scaffold should provide support and space for cells to grow, migrate and adhere and continually retain their phenotype. Hence, scaffolds should be biocompatible and biodegradable in the sense that it is able to propagate appropriate signals for the seeded

Specifically, numerous growth factors have been used on meniscal fibrochondrocytes to evaluate their potential in healing tears or on protein synthesis using cell culture conditions. The most utilized is transforming growth factor-*b* (TGF-*b*). Studies by Marx et al. and Dunsmore et al. showed that TGF-*b* increases

cells execute normal cell homeostasis and processes.

Tissue engineering is deemed as a promising therapeutic tool in treating disease

Tissue Engineering

#### **Chapter 4**

## Bio-Engineered Meniscus for Tissue Engineering

*Azran Azhim, Najian Ibrahim and Fatihah Yusof* 

#### **Abstract**

 Meniscus plays fundamental roles in the knee mechanisms and functions. It acts as a shock absorber where it enables even distribution of forces, and also lubricates knee joints. Meniscal injuries could result to the onset of degenerative osteoarthritis if proper treatments are delayed. To date, treatment of meniscal injuries are more towards conservative methods and surgical approach commonly known as meniscectomy. Attempts to develop scaffolds for meniscus implants from synthetic and biological sources have been done in the recent years. This approach involves a multidisciplinary study known as tissue engineering and regenerative medicine. It involves the combination of three crucial aspects; the choice of chondrogenic/stem cells, bioscaffolds and favourable environmental factors such as growth factors. This chapter discusses and highlights on the currently available meniscal scaffolds that have been explored before. Focus is also directed on the potential of decellularized extracellular matrix (ECM), prepared through sonication treatment that produced scaffolds which mimics natural meniscus. The evaluation of decellularized scaffolds was portrayed through recellularization using cells namely chondrocytes, fibrochondrocytes and stem cells in order to regenerate new functional tissue. In short, this chapter serves as a representation of current approaches aiming in bio-engineering the meniscal scaffolds as meniscus tissue replacement.

**Keywords:** meniscus, bioscaffolds, decellularization, recellularization, implant

#### **1. Introduction**

Tissue engineering is deemed as a promising therapeutic tool in treating disease and injuries. There are three crucial aspects in determining the success of this approach which is by combining cells, biomaterial scaffolds and biologically active molecules such as growth factors. Developing three-dimensional scaffolds or constructs that could serve similarly as native tissue is utterly important. A scaffold should provide support and space for cells to grow, migrate and adhere and continually retain their phenotype. Hence, scaffolds should be biocompatible and biodegradable in the sense that it is able to propagate appropriate signals for the seeded cells execute normal cell homeostasis and processes.

Specifically, numerous growth factors have been used on meniscal fibrochondrocytes to evaluate their potential in healing tears or on protein synthesis using cell culture conditions. The most utilized is transforming growth factor-*b*  (TGF-*b*). Studies by Marx et al. and Dunsmore et al. showed that TGF-*b* increases the proteoglycan synthesis of fibrochondrocytes from all different sections of the meniscus. Besides, hepatocyte growth factor (HGF) or bone-morphogenic protein-2 (BMP-2) was also shown to increase DNA synthesis. To add, cell migration rate improved with the usage of HGF and BMP-2 [1, 2] Other growth factor such as interleukin-1 (IL-1) was also reported to stimulate migration of cells taken from the peripheral third of the tissue. The famous fibroblastic growth factor (FGF) was studies by Webber et al. to show proliferation and stimulate the growth of fibrochondrocytes and human platelet lysate (PL) [3].

 Next, the key aspect in the success of tissue engineering is the development of effective scaffolds which can serve to replace injured or damaged tissue. Thus for a successful meniscus replacement, consideration in the optimal scaffolds properties such as biomechanical, immunogenicity and potential to recellularize cells are important to be scrutinized. The highlight of this chapter would be discoursing different options in developing novel scaffolds of meniscal tissue replacement. The meniscus primarily functions as a load bearer and shock absorber. Degenerative or traumatic loss of meniscal tissue sometimes requires multiple surgical procedures to be treated. Ideally, treatment of meniscal injury should be focusing on the preservation and restoration of the meniscus function. However, effort of complete replacement of meniscus is deemed warranted in more severe cases. Various types of transplant have been done at the experimental and clinical level for example allogenic and autologous meniscus transplant. Issues such as host reactions towards major histocompatibility complexes of the donor, lead to progressive decellularization and consecutive failure of the transplant in meniscus allograft transplantation. To date, none of the proposed replacement methods could provide long-term chondroprotective effect. Scaffolds are necessary for tissue engineered meniscus replacement and considerations such as biomechanical, cell toxicity and immunological response of the host towards the scaffolds are crucial to be scrutinized. There is evidence suggesting that degenerative tears in older patients without mechanical symptoms can be effectively treated non-operatively with a structured physical therapy programme as a first line. Even if these patients later require meniscectomy they will still achieve similar functional outcomes than if they had initially been treated surgically. While, partial meniscectomy is more suitable for symptomatic tears which is hard to repair but could still preserve meniscal function.

#### **2. Composition and cell characteristics of meniscus**

 Meniscus possesses a highly heterogeneous extracellular matrix (ECM) and has a wide range of cell distribution [4]. The ECM of meniscus is categorized by region. Collagen type I accounts for >80% of the composition in the red-red region by dry weight, and the remaining content comprises <1%, including collagen types II, III, IV, VI, and XVIII [5]. There is about 70% of collagen from the dry weight in the white-white region. On the other hand, collagen types II and I account for 60 and 40%, respectively. Next, the cell population in meniscus is categorized into 4 types based on where it resides. First, the outer one-third of the meniscal area is comprised of fibroblast-like cells, demonstrated by elongated shapes while outer periphery contain many cell processes like fibroblasts. Second, the inner two-thirds of the meniscal region mainly contain fibrochondrocytes, oval to round in shape. The inner avascular region comprised more rounded and chondrocyte-like cells. Lastly, fusiform cells are positioned parallel to the meniscal surface at the superficial zone [6].

### **3. Currently available scaffolds for meniscus**

 Meniscus scaffolds serves as a platform for the ingrowth of cells and provide support for the remodeling of the native tissue. There are two categories of scaffolds available; synthetic and biological types. There is a wide variety of synthetic scaffolds that have been explored. Polymer based scaffolds have been tested in a few experimental animal studies. Fibrocartilage-like tissue was able to grow in about 3 months' time after the seeding/implantation [7]. They also reported that in control group, degeneration of hyaline cartilage proceeded slower but could not be halted. To add, other bioabsorbable synthetic polymers, such as polyurethane (PU), polyglycolic acid (PGA), polylactic acid, and poly (- caprolactone) (PCL) are also widely studies to play important part in supporting the development of meniscal scaffolds [8, 9]. The main advantages of using polymer as the main material in scaffolds development is that they provide versatility, comparable biomechanical properties with native tissues and easily available material supply. However, there are some downside of using synthetic polymers which include their hydrophobic properties, non-biocompatibility issues, immunorejection and inflammation. Thus, many attempts have been done to improve polymer based scaffolds. One of them is Koller et al. whom had attempt to enhance the bioactivity of synthetic scaffolds by adding polyethylene terephthalate (PET) to hyaluronic acid/PCL scaffolds and the results were positive [10]. Scaffolds with PET were recorded to express more type II collagen mRNA and secreted more GAGs than without PET. Besides that, Baker and Mauck developed aligned (AL) scaffolds by electrospinning whereby cells in the AL group showed AL morphology whereas those in the control group took a polygonal shape [11]. Koller et al. improved PGA by reinforcing bonding with PLGA at a ratio of 75:25 in order to fabricate meniscus-like scaffolds [10]. Allogenic meniscal cells were seeded into the scaffolds *in vitro* for 1 week to replace the medial meniscus in rabbits. The results showed neomenisci are able to be regenerated which is similar to the native meniscus. However, the newly form neomenisci were not capable to prevent articular cartilage from further degenerating.

They are two main types of natural scaffolds which are tissue derived materials, extra cellular matrix (ECM) components and decellularized tissue. Some of the tissue derived materials that have been studied comprised of small intestine submucosa (SIS), periosteal tissue, and perichondral tissue. Cook et al. have done studies of SIS in dogs and showed promising results. However the study only lasted for 12 week and no mechanical testing was done [12]. One of the significant tests is their major animal trial which consisted of removing 80% of the medial meniscus of the dog and replacing it with this scaffold. The results after 3 years of implantation were promising which showed no degeneration of the articular cartilage. Walsh and co-workers utilized periosteal tissue in rabbits, showed both hyaline cartilage and bone growing in the repair tissue at the end of the 24- week trial. The results from perichondral tissue were not much better; these 12-month sheep tests gave repair tissue that resembled the meniscus grossly, but the tensile modulus of the repair tissue was much lower than native menisci. Besides that, collagen based scaffolds have also been developed from porcine small intestine submucosa (SIS) but however they failed to portray consistent results in experimental animal studies [13, 14].

Next, naturally derived ECM components include collagen, proteoglycans and elastin molecules. These scaffolds were made from collagen retrieved from bovine tendons and then molded into a circumferential orientation. It is now is already in phase II clinical trials. Collagen scaffolds portrayed a more

convincing result whereby Stone, Rodkey, and co-workers used collagen-GAG scaffolds [15].

Attempts in developing decellularized tissues while retaining its ECM properties have been recently studied. Simple tissues as well as complicated organs have been decellularized and decellularization methods have been optimized to completely remove the cellular components while keeping the ECM intact. ECM scaffolds and substrates are very ideal candidates for tissue engineering as it functions in providing supporting materials for cell regulations and functions such as cell survival, proliferation, morphogenesis and differentiation. By comparing these three types of scaffolds, it is undeniable to claim that decellularized based scaffolds which could still contain ECM properties hold the greatest potential in developing ideal scaffolds for meniscus. The next sub topic will be discussed trials to develop decellularized matrices various techniques; biological, chemical and physical methods.

#### **4. Decellularized meniscal scaffolds**

 Decellularized scaffolds are expected to provide a better alternative for implant development in tissue engineering. Besides of it being a suitable microenvironment for cells, it also preserves appropriate meniscal geometry. Nevertheless, some challenges should be addressed to obtain ideal meniscal scaffolds. Because of meniscus natural shape, it will make it tougher for cells to evenly penetrate a decellularized meniscus. Not only that, an abundance of bone morphogenetic protein-2 (BMP-2), a member of the TGF- superfamily will directly stimulate MSC differentiation and can affect cell migration [16]. A study by Minehara et al. used recombinant human bone morphogenetic protein-2 (rhBMP-2) loading in solvent-preserved human menisci to induce migration of chondrocytes into decellularized which successfully induces migration of chondrocytes thus improving proteoglycan production *in vitro*  [17]. Thus, decellularized scaffolds face a challenge of allowing better cell penetration and migration which depends on variety kinds of exogenous chemokines.

One of the effective detergents used to decellularize menisci is SDS whereby collagen structure is retained [18]. Biomechanical testing using repetitive ball indentation test (stiffness, N/mm; residual force, N; relative compression force, N) on the processed tissue were similar to those of the intact meniscus, and the histological results showed no residual cells. Besides that, Maier et al. used a self-developed enzymatic process to treat ovine menisci whereby results suggested that native cells and immunogenic proteins (MHC-1/MHC-2) are completely removed while retaining significant biomechanical traits [19]. On the other side, Stabile et al. attempted to improve the porosity of decellularized scaffolds by applying concomitant decellularization and oxidation processes [20]. Azhim et al. implemented neoteric sonication decellularization system to produce decellularized bovine meniscal scaffolds [21]. These scaffolds provide similar biomechanical properties of native meniscus, and were able to completely remove the immunogenic cell components. However, the sonication treatment compromised the native ECM components and collagen fiber arrangement. Thus, using decellularized scaffolds are great alternative for implants but more improvising needs to be done in order for successful integration into patients.

#### **5. Decellularization strategies**

Biological scaffolds had been widely used in tissue engineering and regenerative medicine field because it virtually resembles native tissue due to the presence

#### *Bio-Engineered Meniscus for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.80553*

 of versatile bioactive nature within the extracellular matrix components [22]. The preparation of natural biological scaffolds involves a process known as decellularization as shown in **Figure 1**. Decellularization is a process that removes whole cellular components within the existing tissue while preserving the composition, integrity and mechanics of the three dimensional extracellular matrix scaffolds to the extent possible [23, 24]. The elimination of the antigens and cellular components from the tissue-derived scaffolds able to reduce the potential immune rejection and inflammation from occurring [25]. The choice of decellularization method varies depending on the characteristics of the particular tissues itself such as geometric considerations, cells and matrix density [26, 18]. An effective and ideal decellularization process supposedly manages to balance the removal of cellular components and preservation of matrix. There are various techniques that had been developed to obtain the most effective outcomes for fabrication of meniscus bioscaffolds using decellularization process. According to Chen & Kawazoe and Gilbert, to obtain an effective decellularization effect, it is encouraged for the method to be applied in combination [22, 26].

 Three main strategies had been performed comprised of biological, chemicals and physical methods. For biological method, it is based on treatments using the enzymes such as proteases (trypsin, dispase), nucleases (DNase & RNase), collagenase, lipase and others [23, 27]. Enzymes are known as substances that have high specificity onto biological substrate which able to cleave or hydrolyze the particular bonds within the tissue structure during decellularization. According to Badylak et al. enzymatic decellularization treatment need a long treatment time and has difficulty to achieve complete cellular components removal alone [28]. Moreover, an extensive treatment time up to 2 days will affecting the ECM ultrastructure components, thus weakening the mechanical properties of the tissues. A study done by Maier et al. treated ovine meniscus with trypsin, collagenase and protease enzyme had successfully decellularized the tissue but with GAGs destruction [19].

The second option is the chemical methods which are further expanded into acid & based treatments, alcohols and also surfactants. According to Seiichi et al., chemical detergents treatment was investigated to be the most commonly used for decellularization technique [29]. The mechanism of acid& bases in decellularizing tissues is by catalyzing hydrolytic degradation of the biomolecules that able to dissociate the DNA from the ECM and disrupting nucleic acid [23, 30]. Chen et al. had performed a decellularization of porcine meniscus using five types of acid consist of acetic acid, formic acid, peracetic acid, succinic acid, malic acid and citric acid with different acid immersion incubation time of 2, 4, 6, 8, 10 and 12 hours [31]. The results portrayed that formic acid with 2 h immersion treatment is the most

**Figure 1.**  *Decellularization and recellularization of meniscus tissue.* 

 effective because it managed to remove almost 96% of the DNA contents with minor adverse effect on ECM collagens and GAGs [31].

Various types of surfactants widely available for decellularization known as ionic, nonionic and zwitterionic detergents. Ionic detergent is recognized as the strongest acting detergent compared to others which are the most broadly applied in decellularization process [27, 32]. Sodium dodecyl sulfate (SDS), Triton X-200 and sodium deoxycholate are the examples of ionic detergents which are the commonly used for various types of tissue. In 2009, Sandmann et al. investigated and published a study on the effect of 2% SDS with 2 weeks incubation time onto biomechanical strength of human meniscus tissues [33]. It was proven that 2% SDS achieved complete cells removal with minor negative impacts on the biomechanical properties of prepared decellularized human meniscus.

 Biological and chemical treatments might result in residual toxicity within the tissues, thus physical treatment for decellularization had been developed. Physical treatments involve freeze–thaw, high hydrostatic pressure, agitation and sonication to disrupt cell membranes and release the cellular components. High hydrostatic pressure system decellularized the tissue by applying pressure from specialized equipment that lead to high cost requirement. This system able to decellularize the tissue in short treatment time but have high risk of extracellular matrix (ECM) ultrastructure disruption due to baric formation of ice crystals throughout the process [34].

In 2010, a novel sonication decellularization with open system had been developed as a new candidate categorized under physical treatment. Sonication system utilizes the ultrasound power assisted by sodium dodecyl sulfate (SDS) to maximize the decellularization efficiency. Researchers and expertise have used ultrasound technology in a wide range of activities such as electrochemistry, food technology, chemical synthesis, material extraction, nanotechnology and surface cleaning [35]. Recently, the application of ultrasound is said to be one of the popular method for cell disruption, emulsification and homogenizing of biological matter [36]. The potential of ultrasound has lead to the development of sonication treatment.

 Since 2010, a pilot study had been started by using aorta [21, 38–42] and meniscus tissues [21, 37, 43, 44] as model by testing using different sonication frequency and different percentage of SDS solution concentration that suit with the characteristics of the tissue [21, 37–41]. As for decellularized meniscus tissue preparation, primary study was done using 20 kHz frequency with 2% SDS solution for 10 hours treatment time that resulted in highest cells removal but there was minor presence of cells observed [37]. Thus, further study was done by increasing the sonication to 40 kHz frequency while minimizing the SDS concentration to 0.1% in order to preserve the bioscaffolds properties [21]. This study compared the sonication treated tissue with immersion treatment as control and native tissue. Based on the result of van Gieson staining portrayed in **Figure 2**, it revealed the complete cells removal from meniscus tissue by sonication system (C) where there is no nuclei stained can be observed compared to control (B) and native (A).

In 2014, Azhim et al. had developed a novel closed sonication decellularization system as shown in **Figure 3**. The ultrasonic transducer is the source of sonication that has three different set of frequency of 40, 120 and 170 kHz. The decellularization efficiency of sonication system was contributed mainly by sonication and also SDS detergent. Firstly, sonication influences the process by the disruption of the cell membrane and cell contents release by its phenomenon of acoustic cavitation. Besides that, sonication also assists the flow of SDS solution that thoroughly penetrates

**Figure 2.** 

*Photographs of van Gieson staining from surface part of native tissue (A), immersion treated tissue (B), sonication treated tissue (C) with 40× magnification. Yellow circle demonstrated the dark blue nuclei stained [21].* 

#### **Figure 3.**

*Sonication decellularization system consists of ultrasonic transducers, pump, cooling water bath, reactor, actuator, temperature monitor, hydrophone, and multiparameter meter that consists of dissolved oxygen (DO), conductivity and pH sensor [39].* 

within tissue sample. Ionic SDS detergents aid in decellularization by solubilizing nuclear cellular membranes and removing cells residues from the tissue specimens.

#### **6. Recellularization strategies for regeneration of engineered meniscus**

In tissue engineering and regenerative medicine, the preparation of engineered meniscus tissue required triad components comprised of scaffolds, cells and growth factors. Basically, the cells will be recellularized onto artificial or natural biological scaffolds with the presence of growth factors for regeneration of tissue. Recellularization using the three dimensional decellularized scaffolds had been one of the attention recently because it resembles natural tissue that have similar biological compositions of the ECM. It is therefore an advantage for the decellularized scaffolds to provide a better environment for the adhesion, differentiation and proliferation of the seeded cells to regenerate functional tissues [45, 46].

According to Chen, recellularization is a crucial part in tissue engineering where cell-seeded constructs can be prepared [31]. This constructs are believed to have many potential advantages for in vitro and in vivo study. First and foremost, it

manages to provide specific microenvironment for cells to proliferate and perform cellular activities for production of ECM. Besides that, cell seeded constructs also ease the integration between scaffolds and native tissues once implanted into the recipient [31].

The advancements of tissue engineering nowadays had the potential to drive meniscus regeneration into clinically relevant strategies and as a promising avenue to improve meniscus repair. For meniscus tissue engineering, various types of cell sources currently being utilized for recellularization process to prepare the cell seeded constructs [47–49]. Different types of cells stimulate different outcome. Ideal cells that suitable for recellularization of meniscus scaffolds should be easy to obtain, low immunogenicity level and able to regenerate the ECM components within the tissue [7]. In this chapter, we will give an overview of promising cell sources that hold great potential meniscus tissue regeneration. There are two main classifications of promising cells available for seeding processes which are stem cells/progenitor cells and mature cells. **Table 1** summarizes the cell sources that are available and broadly applied in meniscus tissue engineering.

#### **6.1 Stem cells/progenitor cells**

Stem cells are known as undifferentiated cells that able to proliferate and differentiate into many specialized cell types. Two main characteristics of stem cells that distinguish them with other cells is that stem cells are multipotent where it can be induced into specific tissue with specific functions and it has long term self-renewal [50, 51]. Mesenchymal stem cells are the most studied stem cells that can be harvested from several musculoskeletal tissues such as bone, bone marrow, adipose tissue, synovial membrane and cartilage [47, 52]. Drawbacks of using mesenchymal stem cells lies in the complex understanding of required stimuli to direct the differentiation process to a desired lineage [48]. Besides that, once the microenvironment changes and undergo hypertrophy, the differentiated phenotype can be easily lost [53].

 Bone marrow mesenchymal stem cells (BM-MSCs) derived from bone marrow compartment with high proliferative activity is identified as heterogeneous population of stem cells capable to undergo self-renewal [54]. The extraction of BM-MSCs is quit complex because need to undergo bone marrow aspiration procedure which is invasive. BM-MSCs have the ability to differentiate into three lineages of skeletal tissue cells in appropriate in vitro condition; osteoblasts, adipocytes and chondrocytes [55]. Few studies had been attempted in meniscus tissue engineering using BM-MSCs. An in vitro study performed by Yamasaki et al., 2 × 105 cells were seeded onto decellularized rat meniscus in 48 well plate, incubated for 1, 2 and 4 weeks the cell-scaffolds constructs were evaluated with few analyses [56]. The results obtained from the analyses revealed that there was sufficient repopulation of BM-MSCs within the scaffolds. It was noted that there was sufficient generation of ECM compositions such as collagen and GAGs over 4 weeks in culture that resembles the content in control group. Evaluation of mechanical integrity for regenerated tissues portrayed similar stiffness with normal meniscus tissue after incubated 2 weeks in vitro culture. Unfortunately, this study lack of in vivo study which will be done in further study.

Adipose stem cells (ASCs) are considered as alternative cell sources that available in emerging tissue regeneration. This ASCs was discovered in the early 2000 that have high self-renewal capacity and capable to differentiate into three different cell lineages known as adipocytes, osteoblast and chondrocytes if subjected to desired stimuli [47]. It was reported that the isolation of ASCs is easier compared to BM-MSc that are commonly isolated from the intrapatellar fat pad of the knee and is not invasive. [57, 58]. An in vitro study using adipose mesenchymal stem

*Bio-Engineered Meniscus for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.80553* 


#### **Table 1.**

*Cells sources for meniscus tissues regeneration.* 

cells seeded on four types of different scaffolds incubated for 3 days had been accomplished by Moradi et al. in 2017 [59]. The cell seeded scaffolds were evaluated for mechanical integrity, biocompatibility and gene expression. The performed real time PCR after 3 weeks culture concluded that ASCs scaffolds had an increase in aggrecan and collagen type II expression compared to control group. For in vivo study, the ASCs scaffolds were implanted into rabbit model for 7 months to discover neomeniscus tissue formation. It was resulted that ASCs scaffolds were found to generate homogenous neomeniscus with poor quality [59].

In 2001, a study conducted by De Bari et al. that characterized the synovial MSCs reported that the cells capable to proliferate extensively and maintained their multilineage differentiation potential in vitro culture [60]. According to Ozeki et al., his study chose synovium MSCs to be seeded onto tendon grafts and assessed

the meniscus regeneration through in vitro and in vivo. It was revealed that the tendon grafts with synovial MSCs succeeded to induce early phase of synovial coverage at the defect site and had better integration with the meniscus defect that promote meniscus regeneration compared to control group [61].

Recently, cartilage progenitor cells (CPCs) represented as new and potential great cell sources available for cartilage and meniscus tissue regeneration. CPCs are basically obtained from the full thickness of mature cartilage. The chondrocytes population was first isolated and need to further undergo differential adhesion to fibronectin process in order to obtain the CPCs [47, 62]. It was identified that CPCs appeared in fibrochondrocytes-like appearance with chondrogenic potential. These CPCs were reviewed to be resistant towards common problems faced by MSCs recognized as terminal differentiation and also hypertrophy [47, 63]. Hypertrophy is a situation where the cells tend to enlarge with increase in cell mass that required more energy [63]**.** According to Williams, CPSs were reported to experience better complex chondrogenesis processes compared to other mesenchymal stem cells [62]. CPCs supplied onto meniscus with tears demonstrated that the cells capable to migrate to the tears site of tears injury while promoting bridging across the site [64]. Unfortunately, the study about CPCS in meniscus tissue engineering is still limited.

 Myoblasts are considered as adult stem cells candidate that have multiple differentiation potentials that capable to differentiate mainly into myocytes, adipocytes and osteocytes [65]. Myoblasts were recognized as easily accessed cells with relatively abundant availability with acute donor site morbidity [66, 67]. Chondrogenic differentiation of myoblasts within PLGA scaffolds was concluded to have high cell yields with rapid cells proliferation. Besides that, it was observed that the biochemical compositions and mechanical strength of the implanted myoblast-scaffolds was similar to native tissue [66].

#### **6.2 Mature cells**

Mature cells are a nonprogenitor cells including chondrocytes, fibrochondrocytes and fibroblasts able to be derived from cartilage, meniscus and dermal tissues. The application of mature cells somehow managed to overcome major limitation of stem cells that mainly facing the hypertrophy occurrence [47].

 As for articular chondrocytes (AC), it is principally derived from articular cartilage and can enzymatically isolate. A recent study was done where chondrocytes were seeded directly onto the layer of decellularized porcine meniscus and incubated for up to 28 days. It was reported that the cells able to proliferate healthily and synthesized the extracellular matrix such as collagen type II and GAG within the scaffolds [68]. Besides that, according to an analysis done by Marsano et al. that seeded the cells onto 3D pellet culture to investigate the growth and post-expansion chondrogenic capacity of chondrocytes [67]. Based on the evaluation done after pellet culture for 2, 4 and 6 weeks, it was indicated that articular chondrocytes resulted in high proliferation rate. This AC generated tissue was found to form abundant GAG and collagen type II in inner avascular region at the same time produced collagen type IV in outer vascular region [67]. There had been two other studies portrayed in **Table 1** that recellularized scaffolds with chondrocytes for meniscus regeneration [69, 70].

Meniscal fibrochondrocytes are investigated as one of the cell sources for meniscus regeneration which can be extracted easily from meniscus [19, 71]. Fibrochondrocytes distributed in all regions of meniscus but dominates more in the vascular outer layer that mainly composed of Type I collagen. The multilineage differentiation particularly favors more towards chondrogenesis and *Bio-Engineered Meniscus for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.80553* 

adipogenesis [72]. A study done by Maier et al. 2007 had successfully seeded the fibrochondrocytes onto the decellularized ovine meniscus. The results showed that the cells managed to infiltrate, survive and proliferate for more than 28 days in the scaffolds [19].

 The red-red region of the meniscus that mainly exhibit collagen type II consist of fibroblast-like cells with elongated morphology [73]. This kind of cells frequently derived from reliable sources such as dermal skin that obtained from different body sites and also meniscus tissues. Fibroblasts play important roles in physiological process essentially in ECM production, inflammation and wound healing regulation [74]. Stapleton et al. had constructed decellularized scaffolds using freeze thaw method and Sodium dodecyl sulfate (SDS) detergent [75]. The prepared scaffolds were utilized for recellularization process using dermal fibroblasts cells for 7 days and proceed with implantation in GTKO mice. Based on the evaluation after 7 days of culture in decellularized meniscus, the fibroblasts successfully infiltrate the scaffolds to a depth of 150 mm and the cells were found to appear on the surface with flattened morphology [75].

#### **7. Future prospects for meniscal tissue engineering**

 There is a dearth in the number of studies done currently in engineering meniscus as compared to other tissues like articular cartilage. The fundamental knowledge on meniscus and its mechanism needs to be fully understood for researchers and clinicians to target a problem. Besides that, knowledge fibrochondrocytes, chondrocytes and other mesenchymal stem cells reactions to a variety of growth factors need to be understood through a variety of tests, both from tissue engineering studies and meniscal repair enhancement studies. Meniscus scaffolds for future application should focus on engineering the entire functional unit which includes meniscal body with anterior and posterior ligaments. Great perception regarding the characteristics of the meniscus constructs in designing for meniscal replacement is important to the overall function. However, an intensive attempt in discovering the appropriate seed cells, biological and mechanical strength stimulation should be given more attention. Broad study on cell sources chose for recellularization should focusing more to in vivo study in animal model. The well performance of in vivo study might depend on several factors such as the quality of neotissue formed during in vitro. More importantly, the fabrication of engineered meniscus tissue with excellent mechanical strength and function that mimic native tissue is the key issue in this field. Good mechanical strength contributes to normal meniscus function. Besides that, three-dimensional construct printing could profit the development of meniscal scaffolds ideally. In short, the development bio-engineering ideal scaffolds for meniscal tissue engineering applications depend on the ability to preserve the biomechanical and biochemical properties of the tissue. The scaffolds should be biocompatible and emit minimum inflammatory effect on to the host.

*Meniscus of the Knee - Function, Pathology and Management* 

### **Author details**

Azran Azhim\*, Najian Ibrahim and Fatihah Yusof Department of Biomedical Sciences, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, Kuantan, Pahang, Malaysia

\*Address all correspondence to: azr2020@gmail.com

© 2018 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.

*Bio-Engineered Meniscus for Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.80553* 

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**94**

### *Edited by Taiceer Abdulwahab and Karl Almqvist*

Te principal aim of this title is to provide the arthroscopic orthopaedic surgeon with a clear, concise account of the anatomy, pathology, conservative and operative surgical techniques in the management of meniscal pathology. Meniscal lesions are extremely common, and arthroscopic meniscal surgery is one of the most common orthopaedic surgical procedures performed. Te art of meniscal surgery involves many steps, with ever-evolving techniques and implants. Tis book has been prepared during a period of widespread debate on, and evolution in, the conservative, surgical, and biological techniques for managing meniscal lesions. Tis text will help consolidate the current evidence to enable the development of optimal management plans for meniscal injuries.

Published in London, UK © 2019 IntechOpen © pavelalexeev / iStock