Evaluating Donor Variability In Osteogenic Potential In Mineralized Collagen Scaffolds

Introduction:: Bone healing varies between individuals due to a multitude of factors including age, pre-existing health disparities, and lifestyle choices. The healing capacity of bone decreases with age due to increased inflammatory cytokine production, reduced macrophage sensitivity, and decreased mesenchymal stem cell (MSC) metabolism. In the field of tissue engineering and regenerative medicine, a wide range of cell types have been used for in vitro studies of osteogenesis including adipose stem cells, bone marrow stem cells, and mesenchymal stem cells however there is a urgent need to evaluate how donor variability influences osteogenesis and other relating regenerative processes. A comprehensive review published in 2021 found that over 96% of biomaterials literature does not report the sex of the cells used let alone any other patient characteristics [1]. This suggests there is an extraordinary opportunity and need to study sex differences of cell-biomaterial and cell-cell interactions. Our lab has developed a class of mineralized collagen scaffolds which have shown increased osteogenic response in vitro and increased mineral formation in vivo. Here, we report donor variability in osteogenic activity within a model mineralized collagen scaffold which has shown promise for enhancing bone repair in craniomaxillofacial defects.

Materials and Methods:: Mineralized collagen-glycosaminoglycan scaffolds were fabricated via lyophilization from a mineralized collagen precursor suspension as previously described. Human mesenchymal stem cells (hMSCs) were purchased from Essent Biologics (adipose tissue derived) or RoosterBio (bone marrow derived) for a total of eight donors, four of each sex. Cells were expanded in culture until confluent and seeded onto mineralized collagen scaffolds for a culture period of 56 days in non-osteogenic growth media. hMSC osteogenic response was evaluated via metabolic activity and cell number, gene expression, protein expression, alkaline phosphatase (ALP) activity, and mineral formation in our scaffolds. All experiments had three or more experimental groups and we considered Donor and Sex as experimental factors. Residuals were tested for normality using the Shapiro-Wilk test, and homogeneity of variance was tested using the Levene Test. A two-way ANOVA followed by Tukey’s HSD mean separation was used for all analyses. If both assumptions were met, we used a standard p-value at p< 0.05. If one or both assumptions were not met, we made our p-value more stringent at p< 0.01.


Results, Conclusions, and Discussions:: The advent of mesenchymal stem cell therapies in regenerative medicine in clinical applications have often led to variable and conflicting results. There has historically been a lack of uniform characterization methods both in vitro and in vivo to understand the sources of MSC potency and identify variations in tissue engineering and regenerative medicine solutions that can enhance their potency. We have developed a model of mineralized collagen scaffolds which has previously shown promise in enhancing bone repair in craniomaxillofacial defects. Our model allows for probing donor variability in MSC response and has the potential in identifying key design parameters that will minimize donor variability while enhancing repair. Over the course of a 56 day culture we observed male donor cells being significantly more metabolically active (Figure 1A) and proliferative compared to female cells. We further observed that metabolic activity and cell number were directly correlated and consistent between donors. Notably, Donor B displayed the highest metabolic activity by day 56 followed by donor G (both male) while Donor H (female) consistently displayed significantly lower metabolic activity and cell number compared to all other donors. Interestingly, female cells displayed significantly more calcium and phosphorus than male cells (Figure 1B). Donors that displayed low metabolic activity and cell numbers exhibited the highest mineral content. Notably, Donor H (female) displayed the highest mineral deposition. Taken together, it appears that mineral content and metabolic activity/cell number are inversely related. Furthermore, we observed no significant differences of MSC secretome between sex; however, there were significant differences between donors. Ongoing work is evaluating gene expression and further proteomic differences between sex and donors. These results point to the complexity within donor variability and the need to determine a standardized and thorough series of metrics to define osteogenic potential for various populations of donors. To design and test tissue engineering and regenerative medicine strategies with clinical relevance it is vital to understand and consider donor cell variability. This work aims to define a standardized workflow to understand this variability in the context of sex, age, and pre-disposed health disparities to better inform strategies for improved regenerative potential.


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