INTRODUCTION: Surgical graft procedures have established a “gold standard” for successful bone repair: osteoconductive scaffold, osteogenic cells and osteoinductive growth factors such as the bone morphogenetic proteins (BMPs). Adopting a viable cell bone allograft requires transfer of living bone to the site of surgery. Following cryoprotection, assets of viability and identity are critical to the composition of the allograft.

METHODS: 4 different lots of cancellous bone matrix that had been formulated for inclusion with DBM fibers as part of a viable allograft product (VIA Form+ (VIVEX Biologics, Miami, USA) were analyzed for cell count, viability, identity, and retention of identity after expansion. Each lot was from a different tissue donor; Median donor age was 56.25 years, (range 50-70 years). Tissue was protected with a polyampholyte cryoprotectant. Cells were removed with 0.3% NB5 collagenase in a-MEM at 3x volume per sample. Culture conditions were α-MEM w/1% PS and 20% FBS, under hypoxia conditions (5% O₂) in osteogenic conditions.

Previous work had isolated cells from cancellous bone replete with marrow, and expanded the cells to evaluate viability and identity following thaw from cryoprotectant. The comparison was made to demonstrate and compare the identity of attached cells and to compare with cells that were enzymatically removed and collected from bone without previously removing the bone marrow before collection.

RESULTS: Cell number per cc of allograft was consistent in empiric mixtures of cancellous bone and demineralized bone fibers. Viability of cells released from the cancellous bone approached 95% and did not differ between the product conformations. Flow cytometry data demonstrated key markers of osteoblast elaboration. Cancellous-derived cultures fulfilled the minimal criteria for MSCs 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 1). Figure 2 demonstrates the cell identity and culture expanded phenotypes. The similarities suggest that bone-derived cells that are viable, and cryoprotected maintain identity to those stripped from the cancellous bone.

DISCUSSION: Viable allograft matrices demonstrate clinical utility that rivals autograft use as a biological adjunct.[1],[2] This study demonstrated that attached cells on cancellous bone scaffold demonstrate osteogenic capacity when removed and cultured for 21 days. Cells which attached to cancellous bone have been used in cell-based viable matrices share a similar phenotype that remains durable in expansion and comparable in identity. Furthermore, the clinical efficacy shown with this product supports appropriate osteoinductive, osteoconductive, and osteogenic potential. Viable bone allograft that demonstrate therapeutic benefit offer an alternative to autograft in spine fusion applications.

[1] Tally WC, Temple HT, Subhawong TY, Ganey T. Transforaminal Lumbar Interbody Fusion With Viable Allograft: 75 Consecutive Cases at 12-Month Follow-up. Int J Spine Surg. 2018;12(1):76-84. Published 2018 Mar 30. doi:10.14444/5013

[1] Tally WC, Temple HT, Burkus JK. Lateral lumbar interbody fusion using a cellular allogeneic bone matrix in the treatment of symptomatic degenerative lumbar disc disease and lumbar spinal instability. Journal of Spine Surgery; September 2021