**3. i-FACTOR™: P-15 peptide**

In the interest of developing products with greater biological specificity and a potentially better safety profile, a number of peptides (rather than proteins like BMPs) have been evaluated for their role and value in bone formation [24]. Peptides differ from proteins in size and structure, typically being much smaller molecules of between 2 and 50 amino acids compared to proteins (e.g., BMPs), which are much larger (>50 amino acids). Many of these peptide sequences are known for having numerous biochemical cellular signaling roles, especially during de novo tissue formation and in remodeling and injury response. Among the more promising peptides are those found in the cell interaction domain of the master control region of Type I collagen [25]. Type I collagen is comprised of two α1 and one β2 polypeptide chains that wrap around each other to form a right-handed triple helix, a collagen monomer. Numerous monomers polymerize to form the massive, rope-like collagen fibrils found in tissues. Type I collagen not only provides a supportive physical scaffold for cells and confers form and strength to tissues including skin, tendons and, in combination with rigid crystalline hydroxyapatite, bones, it also assumes dynamic, biological functions by regulating tissue assembly, cell differentiation, growth, regeneration, and biomineralization. Numerous functional domains and bioactive peptide sequences on a single collagen molecule are present at regular intervals across the width, and along the length of the polymeric collagen fibril. The high density of bioactive sites on collagen makes it the ideal polyvalent substrate for cells and bioactive factors.

**55**

ization nodes [32].

cells.

*Class III Spine Grafts*

*DOI: http://dx.doi.org/10.5772/intechopen.87706*

tion, cell survival, among others.

consequences on cellular differentiation.

genetically programmed downstream cellular responses.

osteoblasts as well as their activity toward bone formation [31].

The importance of these peptides and their functions is highlighted by the fact that they have been conserved over 65 M years of evolution as the cell interaction domain remains the same as that found in dinosaur Type I collagen [26]. This segment of the collagen molecule tends to become exposed and more bioavailable during chemical or traumatic cleavage of the collagen molecule. Among the peptides from the cell interaction domain, one referred to as P-15 was found to be 4500 times more potent for cell binding than the others. This peptide is a 15 amino acid sequence that represents a unique "kinked" tertiary protein structure on Type I collagen that facilitates its presentation to mesenchymal stem cells (MSC's) and their daughter cells along the osteoblastic lineage [27]. P-15 has been found to attract MSC to the implant by providing a favorable environment that facilitates cell attachment. The attraction is followed by a specific receptormediated attachment that activates down-stream molecular events via receptoractivated cascade pathways. These events activate and accelerate new bone formation as they attract, attach, and activate bone forming cells. These processes are circular and self-reinforcing once initiated. In addition, P-15 has been shown to benefit biochemical mechanisms such as proliferation, differentiation, migra-

In 1996, Qian and Bhatnagar published their first investigations on the P-15 peptide for application in bone tissues. In this paper, they showed that attaching this 15 amino acid peptide (P-15) to a calcium phosphate anorganic bone mineral (ABM) led to dramatic increases in cellular response in culture. Their work suggested that this combination might be a useful addition to the bone grafting armamentarium [28]. The in-vitro model demonstrated the ABM-bound P-15 stimulated humanderived pre-osteoblast resulting in significantly increased number of bound cells and the initiation of down-stream molecular events associated with differentiation and osteoinductive activities. Additionally, it was observed that mechanical forces on the cellular cytoskeleton may be generated by P-15 surface integrin interactions. These forces are believed to contribute to mechanotransduction with profound

Over the subsequent decades, numerous in vitro studies demonstrated that the P-15 peptide would elicit specific biological responses from bone forming lineage cells (pre-osteoblasts as well as MSC.) The stimulation/differentiation of MSC was demonstrated at both the molecular level as seen by upregulation of mRNA production, and the protein level as evidence by the cellular release of bone-regeneration associated proteins and growth factors, including alkaline phosphatase, BMP-2 and Collagen Type I [29]. The mechanism of action that elicits these effects is related to the P-15 peptide "plugging in" to surface receptors on these cells, which turns on the

Qian and Bhatnagar showed that P-15 bound to ABM increases the number of bound human fibroblasts and stimulates cellular activation and spreading [28]. Liu et al. demonstrated that P-15 bound to surface increases the number of bound pre-osteoblastic cells and stimulates cellular activation [30]. The authors also noted a significant increase in specific cell surface integrin activation and focal adhesion kinase activation on surface treated with P-15 compared to control substrates, an indication of the direct biological influence of the P-15 peptide on

Yang et al. demonstrated that P-15 bound to ABM stimulates upregulation of cellular BMP-2 and alkaline phosphatase production and the onset of calcification. Alkaline phosphatase production is an indicator of cellular differentiation to

Yuan et al. found that P-15 bound to ABM stimulates early formation of mineral-

#### *Class III Spine Grafts DOI: http://dx.doi.org/10.5772/intechopen.87706*

*Clinical Implementation of Bone Regeneration and Maintenance*

versus the prior approved low dose forms.

Medtronic on March 9, 2011 regarding the optimized AMPLIFY rhBMP-2 Matrix. The FDA nonapproval stemmed from the fourfold increased cancer risks in the investigational group and was linked to the high dose rhBMP-2 form of AMPLIFY

for retrograde ejaculation and urogenital problems after on-label ALIF.

The data synthesis from the YODA initiative and more recent publications report mixed findings with regards to rhBMP-2 usage complications and cancer incidences after rhBMP-2 [20–23]. This same data synthesis suggests that an informed public might have benefited from earlier disclosure and blinded outcome assessment in

In the interest of developing products with greater biological specificity and a potentially better safety profile, a number of peptides (rather than proteins like BMPs) have been evaluated for their role and value in bone formation [24]. Peptides differ from proteins in size and structure, typically being much smaller molecules of between 2 and 50 amino acids compared to proteins (e.g., BMPs), which are much larger (>50 amino acids). Many of these peptide sequences are known for having numerous biochemical cellular signaling roles, especially during de novo tissue formation and in remodeling and injury response. Among the more promising peptides are those found in the cell interaction domain of the master control region of Type I collagen [25]. Type I collagen is comprised of two α1 and one β2 polypeptide chains that wrap around each other to form a right-handed triple helix, a collagen monomer. Numerous monomers polymerize to form the massive, rope-like collagen fibrils found in tissues. Type I collagen not only provides a supportive physical scaffold for cells and confers form and strength to tissues including skin, tendons and, in combination with rigid crystalline hydroxyapatite, bones, it also assumes dynamic, biological functions by regulating tissue assembly, cell differentiation, growth, regeneration, and biomineralization. Numerous functional domains and bioactive peptide sequences on a single collagen molecule are present at regular intervals across the width, and along the length of the polymeric collagen fibril. The high density of bioactive sites on collagen makes it the ideal polyvalent substrate for cells and

Amidst the high-profile controversy, Yale University Open Data Access (YODA) retrieved Medtronic's safety and efficacy data on file in toto. Contract funding support of both the research and preparation of the work was provided by Medtronic to Yale. The Centre for Reviews and Dissemination (CRD) was then commissioned by the YODA initiative in an unprecedented effort by industry to facilitate unbiased review of the relevant benefits and harms of rhBMP-2 as used specifically in spinal fusion surgery; CRD has no direct financial conflict with Medtronic. Two successive publications in the Annals of Internal Medicine were issued in 2013 regarding the findings of the YODA initiative; the dissimilitude between the two publications were the extraction methods and the different studies included [18, 19]. Simmons et al. found rhBMP-2 had increased fusion rates versus ICBG, 12% higher (CI, 2–23%); Fu et al. found similar overall lumbar fusion rates between rhBMP-2 and ICBG Simmons et al. found nonsignificant increased cancer risk after rhBMP-2 (relative risk, 1.98 [CI 0.86–4.54]); Fu et al. found rhBMP-2 at 24 months had increased cancer risk (risk ratio, 3.45 [95% CI, 1.98–6.00]). Fu et al. also found rhBMP-2 to have associated increased risk for wound complications and dysphagia in off-label use in anterior cervical spine surgery, and nonsignificant increased risk

**54**

bioactive factors.

retrospect.

**3. i-FACTOR™: P-15 peptide**

The importance of these peptides and their functions is highlighted by the fact that they have been conserved over 65 M years of evolution as the cell interaction domain remains the same as that found in dinosaur Type I collagen [26]. This segment of the collagen molecule tends to become exposed and more bioavailable during chemical or traumatic cleavage of the collagen molecule. Among the peptides from the cell interaction domain, one referred to as P-15 was found to be 4500 times more potent for cell binding than the others. This peptide is a 15 amino acid sequence that represents a unique "kinked" tertiary protein structure on Type I collagen that facilitates its presentation to mesenchymal stem cells (MSC's) and their daughter cells along the osteoblastic lineage [27]. P-15 has been found to attract MSC to the implant by providing a favorable environment that facilitates cell attachment. The attraction is followed by a specific receptormediated attachment that activates down-stream molecular events via receptoractivated cascade pathways. These events activate and accelerate new bone formation as they attract, attach, and activate bone forming cells. These processes are circular and self-reinforcing once initiated. In addition, P-15 has been shown to benefit biochemical mechanisms such as proliferation, differentiation, migration, cell survival, among others.

In 1996, Qian and Bhatnagar published their first investigations on the P-15 peptide for application in bone tissues. In this paper, they showed that attaching this 15 amino acid peptide (P-15) to a calcium phosphate anorganic bone mineral (ABM) led to dramatic increases in cellular response in culture. Their work suggested that this combination might be a useful addition to the bone grafting armamentarium [28]. The in-vitro model demonstrated the ABM-bound P-15 stimulated humanderived pre-osteoblast resulting in significantly increased number of bound cells and the initiation of down-stream molecular events associated with differentiation and osteoinductive activities. Additionally, it was observed that mechanical forces on the cellular cytoskeleton may be generated by P-15 surface integrin interactions. These forces are believed to contribute to mechanotransduction with profound consequences on cellular differentiation.

Over the subsequent decades, numerous in vitro studies demonstrated that the P-15 peptide would elicit specific biological responses from bone forming lineage cells (pre-osteoblasts as well as MSC.) The stimulation/differentiation of MSC was demonstrated at both the molecular level as seen by upregulation of mRNA production, and the protein level as evidence by the cellular release of bone-regeneration associated proteins and growth factors, including alkaline phosphatase, BMP-2 and Collagen Type I [29]. The mechanism of action that elicits these effects is related to the P-15 peptide "plugging in" to surface receptors on these cells, which turns on the genetically programmed downstream cellular responses.

Qian and Bhatnagar showed that P-15 bound to ABM increases the number of bound human fibroblasts and stimulates cellular activation and spreading [28].

Liu et al. demonstrated that P-15 bound to surface increases the number of bound pre-osteoblastic cells and stimulates cellular activation [30]. The authors also noted a significant increase in specific cell surface integrin activation and focal adhesion kinase activation on surface treated with P-15 compared to control substrates, an indication of the direct biological influence of the P-15 peptide on cells.

Yang et al. demonstrated that P-15 bound to ABM stimulates upregulation of cellular BMP-2 and alkaline phosphatase production and the onset of calcification. Alkaline phosphatase production is an indicator of cellular differentiation to osteoblasts as well as their activity toward bone formation [31].

Yuan et al. found that P-15 bound to ABM stimulates early formation of mineralization nodes [32].

In vivo studies using a rabbit drill hole model demonstrated that ABM-bound P-15 significantly enhances the generation of new bone formation yielding histological evidence of mature bone tissue [33]. The P-15/ABM material yielded statistically more new bone formation at two, four and 8 weeks with over seven-times higher percentage of new bone as compared to ABM alone [33]. A sheep interbody lumbar fusion animal study demonstrated that ABM-bound P-15 yielded fusion rates equivalent to the "gold-standard" of iliac crest bone graft and displayed good trabecular bridging bone structure at 6 months [34]. Finally, rabbit intramuscular implant studies of ABM-bound P-15 established that the P-15 peptide does not support bone formation outside of a bony tissue environment. This can be interpreted as a safety factor, since ectopic bone formation in clinical use is unlikely. These effects translated from tissue culture into animal implantation, showing promise for bone grafting applications with strong bone formation in the absence of ectopic bone formation.

In 1999, the FDA granted the first of two PMA approvals for the use of the P-15 peptide for dental bone grafting to Ceramed on the basis of a prospective, randomized, Level-I IDE study demonstrating safety and effectiveness. This product, Pepgen P-15, has been used in ~500,000 patients to date in the United States.

In 2000, Cerapedics began developing the P-15 peptide technology platform, called i-FACTOR™ bone graft (P-15 Putty), for use in orthopedics and spine surgery indications. i-FACTOR bone graft is a composite bone graft consisting of the synthetic P-15 peptide (biomimetic of the Type I collagen peptide) absorbed onto ABM (naturally-derived calcium phosphate particles) and then suspended in an inert hydrogel carrier. Cerapedics received the first CE mark for i-FACTOR bone graft in 2008 for all orthopedic applications, including spine. Under the CE mark, the product has been used in >50,000 patients to date.

Cerapedics initiated an IDE trial for single level ACDF in an allograft ring in 2006, which culminated in PMA approval in 2015. This FDA-approved trial was prospective, randomized, blinded, controlled and statistically-powered, thus represents Level I study data [35]. In this 319-patient trial, i-FACTOR bone graft successfully met the predefined noninferiority criteria for radiologic fusion (88.9 vs. 85.8% for control), neck disability index (28.8 change vs. 27.4% for control), neurological success (93.7 vs. 93% for control), and safety (97.5 vs. 95.4% for control). More importantly, an FDA-mandated, prospectively designed statistical analysis of the Overall Clinical Success, defined as individual patients who were successful for all four of the primary outcomes, demonstrated statistical superiority to local autograft in overall clinical success (68.8 vs. 56.9%) at 12 months. This statistical superiority was maintained at the 24-month evaluation.

Following the introduction of the i-FACTOR bone graft in the EU, based on a CE-mark, numerous clinical evaluations were performed with i-FACTOR bone graft in the lumbar clinical indication. Mobbs et al. published a prospective ALIF study in which i-FACTOR was used as a stand-alone bone graft inside a PEEK interbody device [36]. In this study, an independent radiological evaluation found a 94% fusion rate by thin cut CT at 24 months, along with a statistical improvement in all clinical evaluations. The authors concluded that, based on their experience, "the study demonstrates a high fusion rate and clinical improvement comparable to the published results for ALIF using autograft or BMP, while avoiding the complications specific to those materials." This study represents an approved use in the EU and Australia, which would be considered off-label in the United States.

Lauweryns et al. published the results from a prospective intra-patient randomized study comparing i-FACTOR bone graft to local autograft in PLIF fusions [37]. In this study, contralateral cages were randomized to be filled with either i-FACTOR bone graft or local autograft, and fusions were assessed by thin cut CT. This study

**57**

worldwide.

**5. Conclusions**

*Class III Spine Grafts*

the United States.

form of i-FACTOR bone graft.

**4. The OP-1 implant: BMP-7**

*DOI: http://dx.doi.org/10.5772/intechopen.87706*

demonstrated faster fusion with i-FACTOR bone graft compared to local autograft. i-FACTOR bone graft was statistically superior with regards to percentage of patients with complete bridging fusion at both 6 months (97.7% for i-FACTOR vs. 59.1% for autograft) and 12 months (97.8% for i-FACTOR vs. 82.2% for autograft). At 24 months, the fusion rates were no longer statistically different. The authors concluded that "i-FACTOR is associated with faster formation of bridging bone when compared to autologous bone in patients undergoing PLIF." This study represents an approved use in the EU and Australia, and would be considered off-label in

In March 2018, the FDA approved an IDE for Cerapedics to initiate another IDE study. This second prospective IDE study is in single level TLIF procedures. In this study, an advanced formulation of P-15 (P-15 L bone graft) is being randomized against local autograft as the control. This study is expected to enroll 364 patients

Both the well-established mechanism of action regarding the stimulatory effects of P-15 peptide along with the extensive clinical data resulting from an IDE, Level I clinical study, strongly support the safety and effectiveness of P-15 peptide in the

There is a third drug-device combination spinal bone graft product, which deserves some discussion: the OP-1 implant formerly commercialized by Stryker Biotech under FDA humanitarian device exemptions (HDE) OP-1 was bone morphogenetic protein (BMP)-7 on a collagen delivery carrier. The BMP-7 was bound to the collagen prior to packaging and terminal sterilization. Following study in long bone nonunions, the OP-1 implant was studied as an autograft replacement for

After failing to meet the primary outcomes of the study to qualify for a PMA approval [rejection at the FDA advisory panel meeting in November, 2007], Stryker Biotech filed an HDE for revision PLF, which was granted in 2004. The Humanitarian Device Exemption (HDE) pathway is a method of gaining very limited FDA approval for a medical device. The device has to be intended to benefit patients in the treatment or diagnosis of a disease or condition that affects or is manifested in not more than 8000 individuals in the United States per year. Although the application is similar to a premarket approval (PMA) application, the product is exempt from effectiveness requirements and, therefore, does not require a wellcontrolled Level I clinical trial. The application is required to only provide sufficient technical information to demonstrate that the device will not expose patients to an unreasonable or significant risk of illness or injury and the probable benefit to health

primary posterolateral spinal fusion (PLF) under an IDE (IDE G990028).

from the use of the device outweighs the risk of injury or illness from its uses.

The OP-1 device was commercialized by Stryker in the United States until 2010 and then, subsequently sold to Olympus. OP-1 was later removed from the market

Nonstructural allograft and cellular allograft products marketed as HCT/Ps do not require any FDA review for safety or efficacy. Synthetic bone grafts and DBM's require a 510(k) for clearance on the basis of animal studies, and most of these technologies have little to no meaningful clinical data. Currently, there are only two

and has a 2-year endpoint for the PMA filing of Level I data.

#### *Class III Spine Grafts DOI: http://dx.doi.org/10.5772/intechopen.87706*

*Clinical Implementation of Bone Regeneration and Maintenance*

the product has been used in >50,000 patients to date.

statistical superiority was maintained at the 24-month evaluation.

and Australia, which would be considered off-label in the United States.

bone formation.

In vivo studies using a rabbit drill hole model demonstrated that ABM-bound P-15 significantly enhances the generation of new bone formation yielding histological evidence of mature bone tissue [33]. The P-15/ABM material yielded statistically more new bone formation at two, four and 8 weeks with over seven-times higher percentage of new bone as compared to ABM alone [33]. A sheep interbody lumbar fusion animal study demonstrated that ABM-bound P-15 yielded fusion rates equivalent to the "gold-standard" of iliac crest bone graft and displayed good trabecular bridging bone structure at 6 months [34]. Finally, rabbit intramuscular implant studies of ABM-bound P-15 established that the P-15 peptide does not support bone formation outside of a bony tissue environment. This can be interpreted as a safety factor, since ectopic bone formation in clinical use is unlikely. These effects translated from tissue culture into animal implantation, showing promise for bone grafting applications with strong bone formation in the absence of ectopic

In 1999, the FDA granted the first of two PMA approvals for the use of the P-15 peptide for dental bone grafting to Ceramed on the basis of a prospective, randomized, Level-I IDE study demonstrating safety and effectiveness. This product, Pepgen P-15, has been used in ~500,000 patients to date in the United States. In 2000, Cerapedics began developing the P-15 peptide technology platform, called i-FACTOR™ bone graft (P-15 Putty), for use in orthopedics and spine surgery indications. i-FACTOR bone graft is a composite bone graft consisting of the synthetic P-15 peptide (biomimetic of the Type I collagen peptide) absorbed onto ABM (naturally-derived calcium phosphate particles) and then suspended in an inert hydrogel carrier. Cerapedics received the first CE mark for i-FACTOR bone graft in 2008 for all orthopedic applications, including spine. Under the CE mark,

Cerapedics initiated an IDE trial for single level ACDF in an allograft ring in 2006, which culminated in PMA approval in 2015. This FDA-approved trial was prospective, randomized, blinded, controlled and statistically-powered, thus represents Level I study data [35]. In this 319-patient trial, i-FACTOR bone graft successfully met the predefined noninferiority criteria for radiologic fusion (88.9 vs. 85.8% for control), neck disability index (28.8 change vs. 27.4% for control), neurological success (93.7 vs. 93% for control), and safety (97.5 vs. 95.4% for control). More importantly, an FDA-mandated, prospectively designed statistical analysis of the Overall Clinical Success, defined as individual patients who were successful for all four of the primary outcomes, demonstrated statistical superiority to local autograft in overall clinical success (68.8 vs. 56.9%) at 12 months. This

Following the introduction of the i-FACTOR bone graft in the EU, based on a CE-mark, numerous clinical evaluations were performed with i-FACTOR bone graft in the lumbar clinical indication. Mobbs et al. published a prospective ALIF study in which i-FACTOR was used as a stand-alone bone graft inside a PEEK interbody device [36]. In this study, an independent radiological evaluation found a 94% fusion rate by thin cut CT at 24 months, along with a statistical improvement in all clinical evaluations. The authors concluded that, based on their experience, "the study demonstrates a high fusion rate and clinical improvement comparable to the published results for ALIF using autograft or BMP, while avoiding the complications specific to those materials." This study represents an approved use in the EU

Lauweryns et al. published the results from a prospective intra-patient randomized study comparing i-FACTOR bone graft to local autograft in PLIF fusions [37]. In this study, contralateral cages were randomized to be filled with either i-FACTOR bone graft or local autograft, and fusions were assessed by thin cut CT. This study

**56**

demonstrated faster fusion with i-FACTOR bone graft compared to local autograft. i-FACTOR bone graft was statistically superior with regards to percentage of patients with complete bridging fusion at both 6 months (97.7% for i-FACTOR vs. 59.1% for autograft) and 12 months (97.8% for i-FACTOR vs. 82.2% for autograft). At 24 months, the fusion rates were no longer statistically different. The authors concluded that "i-FACTOR is associated with faster formation of bridging bone when compared to autologous bone in patients undergoing PLIF." This study represents an approved use in the EU and Australia, and would be considered off-label in the United States.

In March 2018, the FDA approved an IDE for Cerapedics to initiate another IDE study. This second prospective IDE study is in single level TLIF procedures. In this study, an advanced formulation of P-15 (P-15 L bone graft) is being randomized against local autograft as the control. This study is expected to enroll 364 patients and has a 2-year endpoint for the PMA filing of Level I data.

Both the well-established mechanism of action regarding the stimulatory effects of P-15 peptide along with the extensive clinical data resulting from an IDE, Level I clinical study, strongly support the safety and effectiveness of P-15 peptide in the form of i-FACTOR bone graft.
