PRP-Lysate Infused Gelatin Hydrogel as a Scaffold for Bone Reconstruction

Abstract

In implant dentistry, large vertical and horizontal ridge deficiencies are challenges clinicians continue to face. Despite the current advances in grafting materials and techniques used in the clinic, often the reconstructed defect is inadequate to support implants. The complications rate for vertical alveolar ridge augmentation varies from 12% for guided bone regeneration to 24% and 47% for bone blocks and distraction osteogenesis, respectively. One of the problems related to the use of porous calcium phosphate blocks for these applications is the absence of a suitable scaffold/matrix within the pores to enable host osteogenic and vascular cells to migrate into the block, proliferate, and synthesize new bone. For smaller defects, this role is often met by the blood clot which provides a fibrin matrix and platelet-derived growth factors. For porous blocks in large defects, however, a clot does not adequately fill the pores and does not persist long enough to accommodate cell migration into the center of the block. The overall objective of our work was to develop a gelatin-based gel incorporating platelet-rich plasma lysate, to mimic the role that a blood clot would normally play to attract and accommodate the migration of host osteoprogenitor and endothelial cells into the scaffold and thereby facilitating bone reconstruction. A conjugate of gelatin (Gtn) and hydroxyphenyl propionic acid (HPA) was commended for this use because of its ability to undergo enzyme-mediated covalent cross-linking under the control of horseradish peroxidase and hydrogen peroxide. One specific aim of this study was to assess the migration of rat mesenchymal stem cells (MSCs) into Gtn-HPA under the influence of rat platelet rich plasma (PRP) lysate incorporated into the gel. Gels incorporating PDFGF-BB and without growth factor incorporation (blank gels) served as control groups. We hypothesized that the growth factors in PRP lysate would serve as chemoattractants for the MSCs. The second specific aim of this study was to assess the permissibility of the hydrogel of differentiation of MSCs into osteogenic cells. The hypothesis was that the incorporation of PRP lysate into the hydrogel scaffold improves bone regeneration. One of the challenges in incorporating growth factors into biomaterials to improve the host regenerative ability is the duration and sustainability of their release at the site of the defect. The duration of release from many biomaterials is limited to a few days. This period needs to be extended for vertical and horizontal bone reconstruction. A third specific aim was to evaluate the growth factor release profile from the Gtn-HPA. Our hypothesis was that the Gtn-HPA will not only serve as a scaffold but also can act as a delivery vehicle that allows sustained and controlled release of the infused therapeutic agents over extended periods of time. Results: The number of MSCs migrating into the hydrogel was significantly higher in the PRP lysate-incorporating hydrogel group (640±240; mean ±SD) compared to the PDGF-BB (196±81) and the blank gel control groups (160±41); n=6; p-values were 0.0006 and 0.0003, respectively. For the differentiation/osteogenesis assay, the area% of osteocalcin +cells was significantly higher in both the gel/PRP and gel/PDGF-BB groups, compared to the two control groups. where cells in the blank gels were supplemented in one group with cell expansion medium and the other with osteogenic medium. The results of the ELISA release assay indicated that Gtn-HPA acted as an effective delivery vehicle for the sustained release PDGF-BB was from the three experimental groups, the two PRP-lysate batches and the pure PDGF- infused hydrogel, about 58%-64%-98.8% of the original PDGF-BB amount remained respectively in the gel at 28 days. Conclusions: The results of the in vitro experiments supported our 3 hypotheses regarding the utility of Gtn-HPA for bone reconstruction. The data showed that Gtn-HPA was permissive of cell migration. PRP-lysate infused hydrogels displayed increased rat MSC migration into the Gtn-HPA compared to the blank gel. Regarding the differentiation/osteogenesis assay, the PRP-lysate and the PDGF-BB incorporating gel showed a greater area% of osteocalcin+ cells compared to the blank gel containing hydrogel. The release of the growth factors from PRP lysate, incorporated onto the gel, extended over the period of time necessary for bone reconstruction. These findings commend Gtn-HPA incorporating PRP-lysate for infusion into porous calcium phosphate blocks for vertical and horizontal ridge reconstruction.