Serum-Free Expanded Hair Follicle Mesenchymal Stem Cells Promote Cartilage Repair in a Murine Full-Thickness Defect Model

Serum-free expanded hair follicle mesenchymal stem cells (hfMSCs) maintained mesenchymal phenotype and multipotency during static and stirred suspension bioreactor (SSB) expansion and promoted histological cartilage repair in a murine full-thickness cartilage defect model, with minimal direct engraftment yet expressing of a number of secreted protein (in vitro and in vivo), supporting a primarily paracrine mechanism of action. Osteoarthritis is characterized by irreversible cartilage degeneration and limited intrinsic repair, and current treatments largely address symptoms rather than restoring cartilage structure. Mesenchymal stem cell therapies show promise for cartilage repair but are constrained by invasive tissue harvest, donor variability, and the need for scalable, serum-free biomanufacturing approaches. hfMSCs obtained by minimally invasive hair plucking represent an accessible MSC source, but their performance after serum-free static and SSB expansion for cartilage repair has not been fully defined. hfMSCs from 4 adult donors were adapted from fetal bovine serum-containing media into a fully defined serum-free medium (PPRF-MSC-6) and expanded in either 2D static flasks or SSBs using Cultispher G microcarriers under serum-free conditions. Phenotype and potency were assessed by flow cytometry for canonical MSC markers and lack of hematopoietic markers, along with tri-lineage (osteogenic, adipogenic, chondrogenic) differentiation assays and quantitative mass spectrometry-based proteomics with pathway analysis. A NOD SCID murine full-thickness femoral groove cartilage defect model was used; 1 wk postinjury, mice received intra-articular injections of 1 × 10⁵ hfMSCs (static- or SSB-expanded, male or female donor) or phosphate-buffered saline, followed by Safranin O-based histological scoring and immunofluorescent detection of human nuclear antigen and human SOX9 at 4 wk postinjury. Serum-free hfMSC expansion achieved robust proliferation in static culture (∼8- to 12-fold over 5 days) and comparable or greater proliferation in SSBs (∼15-fold in a representative donor), while preserving MSC surface marker expression and tri-lineage differentiation capacity across donors and culture formats. Both static- and SSB-expanded hfMSCs significantly improved histological cartilage repair scores relative to phosphate-buffered saline controls, with regenerated tissue showing Safranin O-positive matrix, restoration of cartilage architecture, and integration with native cartilage. Human cell engraftment within the repair tissue was low with minimal SOX9 co-localization, and proteomic profiling revealed only modest differences between static and SSB conditions, with ZNF703 as the sole protein significantly upregulated. hfMSCs expressed a number of secreted proteins in vitro and in vivo including follistatin and α2-Macroglobulin. hfMSCs derived from plucked hair follicles can be expanded under fully defined serum-free conditions in either static flasks or SSBs while retaining mesenchymal phenotype, multipotency, and cartilage reparative function in vivo. The comparable repair outcomes between static- and SSB-expanded cells, together with minimal proteomic alterations, indicate that scalable serum-free bioreactor processing can generate therapeutically competent hfMSCs suitable for translational cartilage repair applications. Potential donor- and sex-associated variability in proliferation, engraftment patterns, and repair scores highlight the need for donor screening and standardized potency assays, and future studies in more chronic and immunocompetent models are warranted to define long-term efficacy and mechanistic contributions of hfMSC paracrine signaling.