Cellular Bone Allograft: an in-vitro study

BACKGROUND
More than two million graft procedures are performed annually to repair bone defects. Across a spectrum of use in orthopedics, neurosurgery, and dentistry, autograft bone has been established as a “gold standard” for bone replacement for having components recognized as essential for successful bone repair: osteoconductive scaffold, osteogenic cells and osteoinductive growth factors such as the bone morphogenetic proteins (BMPs). The use of allograft bone and viable (cellular) allograft have been long impeded by perceived safety concerns of disease transmission and lack of osteogenic properties. However, current use of allograft bone and viable (cellular) allograft have overcome safety concerns through advanced donor screening, and through refined tissue processing has led to retention of cell viability as an asset of the graft material. Here, we describe an in-vitro analysis of a new Cellular Bone Allograft, demonstrating the presence of progenitor cells capable of migrating, proliferating and have osteogenic differentiation potential.
HYPOTHESIS
Cellular bone allograft contains cells capable of migrating out of the bone matrix and proliferating in an in-vitro environment. Characterization of those expanded cells will demonstrate progenitor phenotype with osteogenic differentiation potential.
METHODS
4 different lots of cancellous bone matrix that had been formulated for inclusion with demineralized bone fibers as part of a viable allograft product (VIA Form+ (VIVEX Biologics, Miami, USA) were selected at random and analyzed for cell count, cell viability, cell identity and retention of cell identity after expansion. Tissue was protected with a polyampholyte cryoprotectant and cancellous bone with existing cell attachment assessed. Culture conditions were α-MEM w/1% penicillin-streptomicin and 20% FBS, under hypoxia conditions (5% O2) for migration and proliferation experiment. Osteogenic differentiation was assessed using Osteogenic media and normoxia conditions for a minimum of 14 days.
RESULTS
Presence of cells capable of migrating out of the bone and forming proliferating colonies was found for all donors tested after 21 days post-thaw. Presence of cells proliferating on bone was also confirmed using a Live/Dead assay by fluorescent microscopy (Figure 1). Cancellous-derived cultures fulfilled the minimal criteria for progenitor cells [1] as evident by the uniform positivity for MSC markers CD73, CD90 and CD105, and the lack of expression for hematopoietic-lineage markers such as CD45 (Figure 2). Alkaline phosphatase staining of expanded cells in osteogenic media demonstrated the differentiation potential of those progenitor cells (Figure 3).
CONCLUSIONS
Viable allograft matrices have demonstrated effective clinical utility in fusion studies that rival autograft use as a biological adjunct.[2],[3] This study has demonstrated that attached cells on cancellous bone scaffold, part of a new viable allograft product, have migration and proliferation capabilities. Analysis of those expanded cells demonstrated progenitor characteristics and osteogenic differentiation potential when cultured in-vitro.