Title: Adherence of Mesenchymal Stem Cells to Commonly Used Orthopaedic Allograft Tissues 

Author: Alexis Cheney 

Specialty: Orthopaedic Surgery

Keywords: mesenchymal stem cells, bone marrow aspirate concentrate, human acellular dermal allograft, osteochondral allograft, cellular adherence, IVIS Spectrum in vivo system imaging

Background: In recent years, biologics have emerged as a viable option for the augmentation of soft tissue healing across a variety of musculoskeletal applications. In particular, the use of bone marrow aspirate concentrate (BMAC) has gained widespread popularity and is used in clinical practice for several orthopaedic indications, including rotator cuff repair and management of focal articular cartilage defects. Mechanistically, BMAC is thought to provide therapeutic benefit by supplying a source of mesenchymal stem cells (MSCs) and growth factors to the site of injury or repair. While they only represent a very small fraction of the total cell population within BMAC, MSCs and the factors they produce are thought to have the capacity to significantly impact musculoskeletal injuries by modulating the biology of healing. Little is known, however, about the practical adhesion capabilities of these MSCs to the biologic surfaces to which they are most commonly applied. Thus, while studies examining outcomes following BMAC administration have demonstrated some promising results, consensus regarding proper application of these biologics is lacking.

Objective: To assess the adherence of MSCs to two commonly used human allograft materials in orthopedic practice: acellular dermal allografts and osteochondral allografts, across varying conditions that mimic current operating room practices, specifically cell-graft incubation time and application of the commercially available cell product adjuvant, fibrin “glue”.  

Methods: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) from a single donor at passage 3 (P3) were stained with CellBrite NIR790 cytoplasmic membrane dye. Human acellular dermal allograft (hADA) measuring 1cm2 and osteochondral allograft (OCA) plugs (10mm in diameter) were placed into separate wells within a sterile 12-well tissue culture plate. A total of 750,000 stained hBM-MSCs were seeded directly onto each sample at a concentration of 30,000 cells/uL. Allografts containing the stained hBM-MSCs were then allowed to incubate for either 10 or 90 minutes, after which they were rinsed three times with sterile phosphate buffered saline (PBS). Experimental groups were defined as those with an incubation period of either 10 minutes or 90 minutes (10m and 90m respectively). In the hADA cohort, a third condition using fibrin glue was evaluated (10m + F). In this experimental group, 20uL thrombin and 20uL sealer protein (commercially available clinical grade product) were added to the hADA immediately following seeding of the stained hBM-MSCs. IVIS Spectrum in vivo system imaging was used to measure fluorescence signal across a series of timepoints. For the 10-minute and 90-minute experimental groups, fluorescence signal was measured and compared both pre- and post-rinse. Fluorescence signal in each well was then subsequently measured at 18h, 24h, and 48h after rinsing three times with PBS. Log10 scale mean fluorescence outcomes were compared by condition and time using a repeated measure (mixed) analysis of variance model. Separate analyses were carried out for each of the two allograft types. A p-value < 0.05 indicated statistical significance. Additionally, to confirm the human MSC phenotype, a small aliquot of the P3 hBM-MSCs used in the cell-seeding experiments was dedicated for flow cytometry evaluation of the following known MSC surface markers CD90, CD29, CD44, and CD105. Positively stained cell populations were compared to unstained cells and fluorescence minus one (FMO) standard controls.  

Results: Separate analyses were conducted among the hADA and OCA samples. For the hADA cohort, comparison of mean fluorescence signal between the 10-minute (10m), 10-minute plus fibrin (10m + F), and 90-minute (90m) treatment groups, demonstrated a statistically significant difference between all three groups after the three sets of PBS rinses. No significant difference was observed across the three groups at the pre-rinse timepoint. The statistically significant difference in fluorescence signal between the 10m and 90m groups as well as the 10m and 10m + F groups persisted at all extended timepoints (18h, 24h, and 48h). However, there was no statistically significant difference in mean fluorescence between the 10m + F and 90m groups at these later timepoints. Our OCA cohort was observed not to have a statistically significant difference between the 10m and 90m treatment groups at any timepoint. Flow cytometry assays confirmed expression of all four of the following known human MSC cell surface markers CD90, CD29, CD44, and CD105, where the positively stained cell population was increased relative to the unstained cell population and fluorescence minus one (FMO) standard controls.  

Conclusions: This current study provides valuable insight into modifiable factors that can impact cellular adherence of hBM-MSCs. Factors such as increased cell-graft incubation time and the use of commercially available fibrin glue were demonstrated to have a statistically significant impact on the adherence of MSCs to different human allograft materials. The observed differences in hBM-MSC fluorescence for different incubation time durations among hADA samples and the lack of difference in fluorescence among human OCA samples may reflect differences in penetration of cell-based products within different matrices of these two distinct graft materials or differences in cell adhesion molecules content. Future investigation is warranted to help elucidate this. However, our current findings provide a starting point for understanding optimal allograft seeding conditions and can be used to guide subsequent in vivo studies as well as enhance evidence-based decision making to inform future treatment practices.