Recent work on HS demonstrated its inherent bent shape and flexibility in solution emanating primarily from the N-acetylated and less highly sulfated regions of the GAG polymer . While overall flexibility of the GAG generally correlated with length, flexibility may be limited regionally. Analysis of the interactions of HS-like polymers with growth factors has demonstrated an intimate interaction between the two, requiring multiple contacts in sulfated regions [28–30]. Similar binding complexity between HSPGs and BMP-2 is expected  and such could be implied from the high binding avidity measured here.
Antibody characterization of the in vitro recombinant with the anti-HS antibodies suggested this rhPln.D1 HS is N-sulfated, within regions of N-acetylation [31, 32], but also that additional sulfation may be limited, as suggested by the lack of interaction with the two heparin-binding mAbs. Further, a proportion of the rhPln.D1 was shown to be expressed with CS, which was immunoreactive with mAb CS-56 , demonstrating the presence of sulfated regions of either type C, D, or A. While CS-56 prefers the octasaccharide sequence of CS-type C-A-D-C or A-A-D-C, additional antibody characterization of the rhPln.D1 GAGs demonstrated no immunoreactivity with MO 225 which also prefers the octasaccharide sequence of CS-type C-A-D-C but reacts only weakly with the sequence A-A-D-C [33, 34]. No immunoreactivity by mAb LY111, which is an antibody thought to prefer the CS-type C-C-A hexasaccharide without the CS-D disaccharride , supports the presence of the disulfated CS-D unit, possibly in the CS-A-A-D-C octasaccharride configuration. Previous characterization suggested a similarity in the rhPln.D1 generated in vitro from HEK 293 cells and from primary endothelial cells  but differences from this GAG characterization expected with in vivo expression remains unclear.
While there is little evidence to support BMP-2 binding of CS, BMP-4 has been shown to interact functionally with a highly sulfated CS  suggesting that both the sulfated HS and CS of the rhPln.D1 core used here could be involved in functional BMP-2 interactions.
While the rhBMP-2 bound tightly to rhPln.D1 that had been immobilized directly in solid phase ELISA, immobilizing the BMP-2 directly to the polystyrene substrate prevented the interaction with soluble rhPln.D1. These data could be interpreted to support the requirement that multiple contacts exist between the BMP-2 ligand and sulfated GAG chains that was discussed above. Direct immobilization of the growth factor to a substrate in a ligand binding assay may limit the freedom of the GAG chains to optimally bind the GF ligand, likely through steric hindrance by the immobilization surface.
Binding avidity between the rhPln.D1 and BMP-2 was found to be significantly tighter than binding to BMP-7. This was not expected because both BMP-2 and BMP-7 primary sequences each contain three conserved triplets of basic amino acids in the N-terminal region near the start of the conserved cysteine knot. The two N-terminal triplets of BMP-7 are, however, more distant from the knot than in BMP-2 and contain one less basic amino acid substituted by Ser . Further, newly reported molecular docking analysis of heparin/HS with BMP-2 demonstrates that non-conserved residues provide additional electropositive binding potential compared to BMP-7 .
Calcium-based ceramic materials, such as hydroxyapatite and tricalcium phosphate, are effective as osteoconductive agents and work well alone as bone void fillers but they are not osteoinductive (as demonstrated above). Tricalcium phosphate has been shown to be a good delivery vehicle for BMP-2, though, and the combination is osteogenic [36–39]. While very high amounts of rhBMP-2 could be adsorbed to the porous, unsintered calcium phosphate apatite used in these experiments, release of the BMP-2 appeared to be rate limiting. Incubating lyophilized particles that had been loaded with varying BMP-2 amounts ranging from 100 μg to 1 mg per ml of TCP yielded similar quantities of BMP-2 released into solution over the first three days, unlike the dose-dependent elution of the pln.247 plasmid that was demonstrated. The fact that BMP-2 release was most rapid during the second day of incubation and decreased thereafter suggested that a TCP volume-dependent pool of “early-release” rhBMP-2 had become established during the loading and lyophilization process. Similar loading and release characteristics were obtained from a non-porous unsintered calcium phosphate apatite although released amounts were several fold higher (data not shown), suggesting that the porous TCP structure utilized in these experiments aided in retention of BMP-2. These kinetics of early and controlled BMP-2 release may have contributed to the efficacy and safety of the technology.
To substantiate specific pro-drug activity of the pln.247 plasmid, we have demonstrated the presence of expression plasmid throughout a 12-week healing period by PCR, and specific mRNA expression of the recombinant core within the wound. However, we have not yet characterized the expressed recombinant within the wound as neither the core nor the associated GAGs can be distinguished from those of native perlecan D1 or other native GAGs with existing tools. These data, therefore, have only indirectly implicated the in vivo role of the recombinant HS or CS in BMP-2 dose-enhancement. While the GAG characteristics of the rhPln.D1 used here were shown to be similar whether expressed from HEK 293 cells or HUVEC , the type and character of GAG can probably be influenced by the cell type and the environment in which it is expressed , although this has not been systematically described in a recombinant system yet. Nonetheless, an analysis of the structure/function relationship, both in vitro and in vivo, is important to pursue.
The B-247 bioactivated TCP appeared to stimulate intramembranous bone formation that was juxta-proximal to the particles, (see ), and new bone that was contiguous with the maxilla. There was no evidence of ectopic bone in this model; the B-247 particles were associated with the development of mature woven bone on top of, and integrated with, the maxillary bone. However, not all B-247 particles were associated with new bone development; in almost every case the most peripheral particles surrounding the developing new bone remained unassociated with new bone formation. This observation might be partly attributable to a natural physiological limitation to volume-increase of the maxillary ridge and a physiologically excessive volume of particles administered throughout the trials. Alternatively, the process of de novo bone formation around the particles is expansive resulting in less particle density within the new bone while pushing the remaining unincorporated particles peripherally. It is likely that a combination of these two phenomena resulted in a border of some unincorporated particles. The relative proportion of unincorporated particles was ultimately a function of the osteogenic capacity of the treatment condition – non-osteogenic TCP conditions resulted in up to 100% unincorporation of particles into new bone, while the most osteogenic conditions resulted in the majority of particles being incorporated into new bone. Limitation of new bone to regions of bioactivated TCP deposition might also be interpreted as a function of the highly retentive nature of the particles for the biologics, in part.
The pln.247 plasmid delivered alone on the tricalcium phosphate particles was not osteogenic. Further, neither non-demineralized human allograft nor very low doses of rhBMP-2 on the tricalcium phosphate particles (<30 μg/ml) were osteogenic with or without the pln.247 plasmid (data not shown). It was apparent, therefore, that the pln.247 plasmid was not osteogenic when delivered alone, though not surprising since osteogenesis directly attributable to augmenting only HS/CS proteoglycan levels has never been reported.
The model using retired breeders is not common and was chosen with reason. Declining stem cell populations and decreased healing potential in these older animals would theoretically limit the rate and extent of bone regeneration as compared to what could be expected in young mammals. Given that these data have direct bearing on repair or augmentation of the jaw, and the majority of those needing oral or periodontal reconstruction are not young, the use of older animals was deliberate so as to challenge the technology as it may eventually be challenged in the clinic.
A body of evidence exists in the literature demonstrating a role for HSPGs, and perlecan in particular, in cell adhesion, cell proliferation, cell differentiation, and cell migration, all of which are crucial to osteoinduction and wound repair. As cited herein, CSPGs can also have a similar role. Despite this existing evidence for HSPGs and CSPGs as potentially valuable adjuncts for a variety of tissue engineering and wound-healing strategies that are mediated through growth factor activities, no HS or CS augmented therapies yet exist.
By therapeutically delivering the cDNA encoding the proteoglycan core sequence, this technology may overcome complications in HSPG or CSPG manufacturing with pharmaceutical consistency. The technology allows site-specificity and tissue specificity of the post-translational HS or CS GAG modifications that would be difficult to predict and provide pharmaceutically. The FDA has designated the B-247 TCP particles as a combination product, under the regulation of CBER, which is a first for bone graft devices, as all others are currently regulated as devices in CDRH.