Minimally Manipulative Method for the Expansion of Human Bone Marrow Mesenchymal Stem Cells to Treat Osseous Defects

Abstract

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

Vancomycin Containing PDLLA and PLGA/β-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells

“…Since these cells had osteoblast-like properties, it was expected that these cells showed a higher ALP activity than hBM-MSCs (43). Our findings were in line with previous studies (44,45).…”

Assessment of stem cell viability in the initial healing period in rabbits with a cranial bone defect according to the type and form of scaffold

“…In this regard, higher cell viability and survival may have beneficial effects. In this study, the stem cells were incubated for 1 hour before transplantation to allow cells to adhere to the scaffold [3334].…”

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

“…Furthermore, 3D cultures of adult human liver stem cells produced islet-like structures and were able to reverse hyperglycemia in mice with severe diabetes combined with immunodeficiency (34). In addition, 3D spheroid cultures allow for the fabrication of bone marrow mesenchymal cells, which retain osteogenic differentiation potential over a monolayer culture of bone marrow mesenchymal cells without the requirement to use chemicals or hormonal modulation (36). Spheroids of mesenchymal stem cells also expressed higher transcription factors that regulate stemness compared with monolayer cultures, along with higher alkaline phosphatase activity and enhanced expression of osteogenesis-associated genes (37).…”