Evaluation of Human Bone Marrow Mesenchymal Stromal Cell (MSC) Functions on a Biomorphic Rattan-Wood-Derived Scaffold: A Comparison between Cultured and Uncultured MSCs

Abstract

The reconstruction of large bone defects requires the use of biocompatible osteoconductive scaffolds. These scaffolds are often loaded with the patient’s own bone marrow (BM) cells to facilitate osteoinductivity and biological potency. Scaffolds that are naturally sourced and fabricated through biomorphic transitions of rattan wood (B-HA scaffolds) offer a unique advantage of higher mechanical strength and bioactivity. In this study, we investigated the ability of a biomorphic B-HA scaffold (B-HA) to support the attachment, survival and gene expression profile of human uncultured BM-derived mesenchymal stromal cells (BMSCs, n = 6) and culture expanded MSCs (cMSCs, n = 7) in comparison to a sintered, porous HA scaffold (S-HA). B-HA scaffolds supported BMSC attachment (average 98%) and their survival up to 4 weeks in culture. Flow cytometry confirmed the phenotype of cMSCs on the scaffolds. Gene expression indicated clear segregation between cMSCs and BMSCs with MSC osteogenesis- and adipogenesis-related genes including RUNX2, PPARγ, ALP and FABP4 being higher expressed in BMSCs. These data indicated a unique transcriptional signature of BMSCs that was distinct from that of cMSCs regardless of the type of scaffold or time in culture. There was no statistical difference in the expression of osteogenic genes in BMSCs or cMSCs in B-HA compared to S-HA. VEGF release from cMSCs co-cultured with human endothelial cells (n = 4) on B-HA scaffolds suggested significantly higher supernatant concentration with endothelial cells on day 14. This indicated a potential mechanism for providing vasculature to the repair area when such scaffolds are used for treating large bone defects.

Bone defect filling with a novel rattan-wood based not-sintered hydroxyapatite and beta-tricalcium phosphate material (b.Bone™) after tricortical bone graft harvesting – A consecutive clinical case series of 9 patients

“…Consequently, such novel rattan-wood based not-sintered hydroxyapatite and beta-tricalcium phosphate material has been launched to the market (b.Bone™, GreenBone ORTHO S.p.A Faenza, Italy). Experimental studies have already confirmed a high level of structural mimicry by the scaffold and showed strongly enhanced osteogenic potential in vitro [9] , [10] , [11] . However, only one case with the implantation of this scaffold into the iliac crest [12] has been published, but clinical data on the safety and performance of the rattan-wood-derived scaffold within larger series have not been reported so far.…”

Fabrication and Characterisation of the Cytotoxic and Antibacterial Properties of Chitosan-Cerium Oxide Porous Scaffolds

“…Ethical approval for the collection of samples was obtained from NREC Yorkshire and Humberside National Research Ethics Committee (number 06/Q1206/127). Bone marrow mesenchymal stem cells (BM-MSCs) were obtained from three healthy donors after informed written consent and processed to obtain mononuclear cells that were culture expanded to isolate BM-MSCs as previously described [ 26 ]. The cells expressed the MSC phenotype of CD105, CD73, and CD90 and were negative for CD45 [ 27 ].…”

In Vitro Osteogenesis Study of Shell Nacre Cement with Older and Young Donor Bone Marrow Mesenchymal Stem/Stromal Cells

“…Pooled young and older donor MSCs (1 × 10 6 cells per sample) were separately seeded on duplicate SNC scaffolds, as described in 2.6.4. At the end of the culture, the SNC samples were processed for gene expression analysis, as previously described [47]. In brief, the samples were treated with 350 µL of Buffer RL to lyse the cells; strong agitation using vortex over ice was used to ensure the removal of all the cells with their genetic contents and to prevent RNA degradation.…”

“…In brief, the samples were treated with 350 µL of Buffer RL to lyse the cells; strong agitation using vortex over ice was used to ensure the removal of all the cells with their genetic contents and to prevent RNA degradation. RNA was extracted according to the manufacturer's protocol (Total RNA purification kit, Norgen Biotek, Canada), reverse transcription was performed using RT master mix (Fluidigm) and the cDNA was pre-amplified with the pooled Taqman assays, as previously described [47,53,56]. Gene expression was performed for genes related to MSC osteogenesis (day 14 samples) and cell cycle regulation (day 7 samples), including bone morphogenetic protein 2 (BMP2), RUNT-related transcription factor X2 (RUNX2), alkaline phosphatase (ALP), collagen type I Alpha 1 chain (COL1A1), Osteomodulin (OMD), secreted protein acidic and rich in cysteine (SPARC), and p16, p21 and p53, respectively.…”

“…As it has been reported that, during aging, BM-MSCs exhibit a shift towards adipogenesis rather than osteogenesis [14][15][16][17]20,45], and given that more than 50% of orthopaedic procedures are performed on older patients [46], evaluating the osteogenic potential of SNC with BM-MSCs from older donors is essential. Previous in vitro osteogenesis studies of other biomaterials such as PCL nanofibrous mat, graphene-incorporated methacrylate gelatin, and rattan wood have been conducted with BM-MSCs from older donors [47][48][49]. However, none of these studies have investigated the senescence status of the older donor BM-MSCs prior to scaffold seeding.…”