Injectable Bioactive Gelatin-Hyaluronan-Calcium Phosphate (GH-CP) and Its Osteogenic Potential for Flapless Guided Bone Regeneration (GBR)
DMSc candidate: Young Kwang Kim, DMD
Submitted to the Faculty of Medicine on May 2019 in partial fulfillment of the requirements for the degree of Doctor of Medical Sciences in Harvard School of Dental Medicine
In this contemporary era of minimally invasive surgery, injectable biomaterial scaffolds have shown their strategic potential in bone tissue engineering (BTE). Completely injectable hydrogel substratum with micro-sized bone graft particulates through a needle-shape orifice permits a new paradigm of flapless guided bone regeneration (GBR) utilizing a tunneled subperiosteal pouch concept. Although several biopolymers and their BTE applications have emerged, there is still a need for a clinically applicable delivery system; a systematic characterization of physicochemical properties; a mechanistic understanding of osteogenic cells within this biopolymer complex; and implementation of chemoattractants, all of which to construct a foundational framework for future in vivo investigations.
The first objective of this thesis is to design and to establish a novel injectable dual-syringe auto-mix system for providing a clinically relevant in situ cross-linking biopolymer hydrogel infused with osteoconductive particles. A combination of gelatin-hydroxyphenyl propionic acid (Gtn-HPA or G gel), hyaluronic acid-tyramine (HA-Tyr or H gel), the covalent cross-linking modulators of horseradish peroxidase and hydrogen peroxide, and calcium phosphate particles with different sizes (~250µm, ~175µm, and ~100µm) were investigated, with respect to content distribution, storage/compressive modulus, and swelling/expansion ratios. Another objective was to evaluate the behavior of bone marrow-derived mesenchymal stem cells (bMSCs) in the gel, including: cellular viability and morphology; proliferation (Ki67); contractility (α-smooth muscle actin, α-SMA); differentiation (osteocalcin/osteopontin, OCN/OPN); and mineralization (Alizarin Red S, ARS, staining). This work included an assessment of the chemotactic and osteogenic roles of bMSCs via incorporating rhPDGF-BB and rhFGF-2. Additionally, a translational cone beam computed tomography (CBCT) volumetric experiment of GBR in an ex vivo model was employed to investigate the structural integrity of the graft-host union.
Our dual-syringe auto-mix system of Gelatin-Hyaluronan-Calcium Phosphate (GH-CP) achieved an immediate in situ cross-linking with microscopically well-dispersed CP particles. This mechanically fine-tunable GH-CP demonstrated higher storage and compressive modulus, and reduced swelling and expansion ratios, compared to gelatin (G) and gelatin-hyaluronan (GH) gels. For viability, G and GH gels revealed highly favorable state, while H gel showed inferiority. Each gel illustrated different morphological traits of bMSCs on the G gel as mostly elongated; the H gel as mostly rounded; and the GH gel as half mixed. With no apparent differences among the three gel groups, α-SMA showed a prominent expression compared to Ki67 levels. Interestingly, GH-CP gel, as expressed with both intra- and extra-cellularly, resulted in a greater osteogenic potential of OCN/OPN levels and ARS staining compared to G and GH gels. It was also demonstrated that pre-soaking of CP particles with the chemoattractants, rhPDGF-BB/rhFGF-2, results in attachment of bMSCs, with the possibility of contact osteogenesis. The culture medium also exhibited notable effects on this in vitro differentiation capability. Along with the aforementioned cellular outcomes, our ex vivo volumetric CBCT analysis also revealed structural integrity of GH-CP over the conventional calcium phosphate. Consequently, these findings provide in vitro physicochemical and osteogenic support for GH-CP to be utilized in such flapless GBR surgical arena with its further potentials in incorporating diverse pharmacotherapeutics.
Thesis mentor: Myron Spector, PhD
Title: Professor of Orthopedic Surgery (Biomaterials), Harvard Medical School
Senior Lecturer, Massachusetts Institute of Technology
Director, Tissue Engineering and Regenerative Medicine Lab, VA Boston Healthcare System||