Purpose

Effective surgical treatment of meniscus injuries has been a challenge for decades. Stimulating meniscus regeneration through transplantation of meniscal progenitor cells has been proposed as a promising new strategy. The purpose of this study was to comprehensively phenotype regional progenitor populations in the human meniscus and donor-matched articular cartilage.

Methods and Materials

Progenitor populations were isolated by selective adhesion to fibronectin from avascular and vascular regions of menisci and articular cartilage (n=5) harvested from human arthroplasty donors, as well as donor-matched whole mixed populations (no selective adhesion to fibronectin) for comparison. Growth kinetics and immunoprofiling were assessed at passage 0, and gene expression analysis and 3D pellet chondrogenic capacity were assessed at passage 2 for the different cell populations.

Results

Fibronectin-selection resulted in populations resembling progenitors from the avascular and vascular menisci (PAvas and PVas, respectively) and also articular cartilage (PChs); these all demonstrated colony forming capacity on monolayer culture, whereas their mixed populations did not. PVas had a significant lower population doubling time cf. their vascular mixed counterparts. PVas also proliferated significantly faster than PAvas and PChs. Each progenitor cell population were significantly more immunopositivite for CD49b and CD49c cf. their mixed populations and PChs had a higher positivity level of CD166 compare to mixed chondrocytes. Collagen type I was upregulated and type II downregulated in progenitor pellets cf. mixed pellets. GAG/DNA analysis demonstrated that progenitor cells produced more GAGs per cell than mixed populations in all regions.

Conclusion

Our study demonstrates that the human meniscus contains meniscal progenitor populations that retain colony forming capacity and have a distinct immunoprofile and matrix forming capacity in both the avascular and vascular regions. The findings of this study build on the body of evidence which suggests that meniscal progenitors represent attractive novel cell therapy populations for the enhancement of meniscal regeneration.

Purpose

The purpose of the study was to evaluate the effects of mesenchymal stem cell-extracellular vesicles (MSC-EVs) on chondrocyte proliferation in vitro and on cartilage repair in vivo following bone marrow stimulation (BMS) of focal chondral defects of the knee.

Methods and Materials

Six adult Göttingen minipigs received two chondral defects in each knee. The pigs were randomized to treatment with either BMS combined with MSC-EVs or BMS combined with phosphate-buffered saline (PBS). Intra-articular injections with MSC-EVs or PBS were performed immediately after closure of the surgical incisions, and at 2 and 4 weeks post-operatively. Repair was evaluated after 6 months with gross examination, histology, histomorphometry, immunohistochemistry, and micro-computed tomography (µCT) of the trabecular bone beneath the defect.

Results

More bone was seen in the cartilage defect area in defects treated with MSC-EVs compared with PBS-treated defects (7.9% vs. 1.5%, p = 0.02). Hyaline cartilage represented less than one percent of the repair tissue in both groups. Using ICRS II histological scoring, defects treated with MSC-EVs scored lower on “matrix staining” (20.8 vs. 50.0, p = 0.03), “cell morphology” (35.4 vs. 53.8, p = 0.04), and “overall assessment” (30.8 vs. 52.9, p = 0.03). In addition, defects treated with MSC-EVs had lower collagen II and higher collagen I areal deposition. The subchondral bone had significantly higher tissue mineral densities in defects treated with MSC-EVs compared with PBS-treated defects (860 mg HA/cm³ vs. 838 mg HA/cm³, p = 0.02).

Conclusion

Intra-articular injections of MSC-EVs in conjunction with BMS led to osseous ingrowth that impaired optimal cartilage repair, while enhancing subchondral bone healing.

Purpose

Osteoarthritis (OA) is a joint disorder causing articular cartilage degeneration. Currently, treatments are mainly pain- and symptom-modifying, rather than disease-modifying. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism-based approach for the treatment of OA. Many studies demonstrate that MSCs attend to tissue repair through the secretion of trophic factors or extracellular vesicles (EVs). We developed a “donor-to-patient” system for aseptic therapeutic cell manufacturing using a xeno-free medium. We validated the potential therapeutic benefits of secreted EVs isolated from BMSC culture in this innovative culture system, for cartilage repair.

Methods and Materials

We characterized (EVs) derived from hBMSCs, grown in a xeno-free culture system (XFS) compared to a conventional fetal bovine serum (FBS) culture system, in normoxic and hypoxic culture setting. We investigated also the therapeutic potential of EVs in an in vitro model of OA. We characterized the miRNA content of EVs in different culture setting to select putative miRNA that could be involved in a biological function.

Results

The biological effects of XFS- and FBS-cultured hBMSC-derived EVs were tested on IL-1α treated hACs in an experiment designed to mimic the OA environment. We observed that under inflammatory conditions hACs are able to recruit and internalize more MSC-derived EVs, especially those derived from cells cultured in our XFS system and in hypoxic conditions. XFS-EVs both in normoxia and hypoxia shows anti-inflammatory properties in an in vitro OA model. Analysis of miRNA content showed upregulation in XFS-hBMSC-derived EVs of miRNAs known to have a chondroprotective role, and also appear to be involved in cartilage homeostasis and influence TGF-beta signaling.

Conclusion

XFS medium was found to be suitable for isolation and expansion of hBMSCs with increased production of EVs. The application of these EVs overcomes the safety concerns associated with serum-containing media and makes ready-to-use clinical cartilage therapies more accessible.

Purpose

Nanotechnology is increasingly empowering the design of innovative methods for cartilage regeneration. Nanomagnetic materials can be used as versatile platforms for drug and bioactive molecules delivery, mechanical stimulation as well as for surface topography modification for the purpose of enhancing stem cell chondrogenesis. We report about the use of magnetic nanoparticles (MNPs) and magnetic nanowires (NW) and their role in adipose derived mesenchymal cells (ADSC) and Wharton Jelly derived mesenchymal stem cells (WJMSCs) chondrogenic conversion in vitro.

Methods and Materials

Human primary ADSCs and WHMSCs were loaded with proprietary bare iron oxide magnetic MNPs sized 10-50 nm by cell particle contact in culture media within a defined interval. Cell viability, proliferation as well as tri lineage differentiation (osteo, adipo and chondrogenesis) were performed. Chondrogenic pellets of ADSC and WJMSC loaded with MNPs were submitted to magnetomechanical stimulation within alternating magnetic field. Pellet histology and the amount of glycosaminoglycan deposition per cell was used to assess chondrogenic conversion. Cell viability and adherence on proprietary magnetic Nichel nanowires (Ni NW) was further tested. Chondrogenic assays were performed in suspension, pellet or in high density culture deposited on NW surfaces.

Results

Both ADSCs and WHMSCs displayed excellent viability and proliferative capability retaining their stemness compared to non-loaded controls. Significantly increased chondrogenesis was displayed by ADSCS-MNP under MF exposure but not by WJMSCs-MNPs. Cells were able to preserve viability in contact with MW, adhered at a relatively low rate on NW surfaces and were able to promote both cell type chondrogenesis.

Conclusion

Nanomagnetic platforms display attractive capabilities in improving ADSC chondrogenic conversion in vitro. MNPs and NW can be used to remotely control cell fate counting for future strategies of cartilage engineering

Acknowledgements

Financial support by the MCID NUCLEU Program (PN 19 28 01 01) and UEFISCDI Contract no. PCE20/2021 (PN-III-P4-ID-PCE-2020-2381) is gratefully acknowledged.

Purpose

Exit from multipotency and lineage commitment of mesenchymal stroma cells (MSC) depends on microenvironmental cues from the stem-cell niche but steering cell-fate into the desired lineage in vivo remains a challenge. Increasing evidence suggests that glycosaminoglycans can be used to activate or sequester growth factors with the specific action depending on sulfation levels. We postulated that differentially sulfated biomaterials can aid developmental lineage instruction of MSC to guide tissue morphogenesis in vivo.

Methods and Materials

Injectable TGFβ-loaded-hydrogels designed at selected different sulfation status of the covalently coupled glycosaminoglycan, to grow true articular-cartilage-like tissue from MSC in vivo, were implanted into subcutaneous pouches of immunodeficient mice. The chondrogenic differentiation, hypertrophy and mineralization were investigated on day-28 and 56 explants by histology, ELISA, qPCR, WB and µCT analysis.

Results

By application of a new injectable TGFβ-loaded-hydrogel we here gained the ability to control skeletal stem-cell fate in vivo down the chondral versus the endochondral pathway depending on the sulfation status of the covalently coupled glycosaminoglycan. High sulfation allowed for long-term TGFβ-retention and silencing of Hedgehog-, BMP- and WNT-pathways and installed pro-chondrogenic and anti-hypertrophic cues in MSC. This permitted in vivo growth of permanent, collagen-type-II-rich neocartilage with long-term resistance to calcification and bone formation. Reduction of sulfation supported Hedgehog/BMP/WNT-signaling switching lineage commitment into endochondral differentiation with strong hypertrophic/osteogenic marker expression, tissue calcification and bone formation.

Conclusion

Our work identifies glycosaminoglycan sulfation as crucial niche instruction signal to determine the chondral stem-cell fate via silencing of prohypertrophic growth factor pathways providing the first proof-of-principle how glycosaminoglycan modification-patterns can determine cell lineage-choice during tissue morphogenesis in vivo.

Purpose

We had previously reported the efficacy of human mesenchymal stem cell (MSC) exosomes in repair of critical-size osteochondral defects in rats and rabbits. To enable clinical translation of MSC exosomes, we proposed a validation of the efficacy of MSC exosomes in a large animal model.

Methods and Materials

Bilateral osteochondral defects (6mm diameter and 1mm depth) were surgically created on the medial femoral condyles of 24 knees in 12 micropigs. Immediately after surgery and at days 8 and 15 post-surgery, 6 micropigs in exosome/HA group received sequential administration of 1mg exosomes in 1ml phosphate-buffered saline (PBS) followed by 1ml hyaluronic acid (HA; Synvisc®) in both knees, whereas the other 6 micropigs in the HA group received 1ml of PBS followed by 1ml HA in both knees. Except for MRI performed on day 15, 2 and 4 months, macroscopic, histological, biomechanical, and micro-CT assessments were performed at 4 months.

Results

At 4 months, exosome/HA-treated defects had significantly higher MRI scores than that for HA-treated defects at day 15 (4.46 vs 3.63; P=0.017), 2 months (7.83 vs 5.79; P=0.023) and 4 months (9.25 vs 6.71; P=0.024). Exosome/HA-treated defects also had significantly better ICRS macroscopic score (9.22 vs 7.25; P=0.008) and ICRS II histological score (79.71 vs 65.10; P=0.032) than HA-treated defects. The mean Young’s moduli of exosome/HA-treated defects were higher than that of HA-treated defects in the defect periphery (19.92 vs 5.50MPa; P=0.003) but modestly in the defect centre (15.17 vs 3.53MPa; P=0.119). Micro-CT analysis revealed structural improvements in the subchondral bone with significantly higher BV/TV and Tb.Th in exosome/HA-treated defects than in HA-treated defects. Importantly, no adverse responses or systemic alterations were observed.

Conclusion

MSC exosomes and HA combination administered at a clinically acceptable frequency of three intra-articular injections promote osteochondral repair with significantly improved morphological, histological, and biomechanical outcomes in a clinically relevant porcine model.

Purpose

We present a preclinical study of use of the Bone Marrow derived Mesenchymal Stem Cells in treatment of ACL tears and cartilage lesions on animal model.

Aims: To assess the technical aspects of the procedure of injection of the BM MSC’s suspended on the fibrin glue; to assess safety of MSC’s application for ACL and cartilage repair and the potential influence of MSC’s on ACL and cartilage healing by hstological analysis.

Presented paper is a part of EXPLORE ME project sponsored by National Center of Research and Development about the use of MSC’s in regenerative medicine.

Methods and Materials

Six animals (12 knees) were used. The animals were divided into the ACL (3) and cartilage (3) groups. In the ACL group iatrogenic damage of the ACL by its detachment at femoral insertion and reinsertion with Internal Brace technique during arthroscopic procedure were performed bilaterally. ACLs in all the right knees were injected with pure fibrin glue. ACLs in the left knees were injected with fibrin glue mixed with 5 x 10⁶ expanded, autologous BM MSC’s.

In the cartilage group, iatrogenic damages of the cartilage at the weightbearing surface of the medial femoral condyle were created. The lesions were filled with hyaluronic scaffold injected with fibrin glue in right knees and with fibrin glue mixed with 5 x 10⁶ expanded, autologous BM MSCs in the left knees in arthroscopic procedures bilaterally.

The animals were euthanised after three months follow up.

Results

Angiogenesis was observed in ACLs MSC+ comparing to control group. Angiogenesis was observed in cartilage samples from MSC-treated group, comparing to controls.

Conclusion

Injection of the MSC’s suspended in fibrin glue is safe and simple procedure. The cells stay inside treated structure. The finding of angiogenesis in treated cartilage wasn’t previously described in literature and requires further investigation.

Purpose

Knee osteoarthritis (OA) represents a common problem with a high social impact, and a significant number of patients complain of bilateral symptomatic OA. The aim of this study was to analyze the preliminary short-term results of a double-blind randomized self-controlled trial comparing intra-articular injection of Adipose Derived-Mesenchymal Stromal Cells (ADSCs) versus placebo for bilateral knee OA.

Methods and Materials

The study design includes 100 patients with bilateral symptomatic knee OA. Each patient receives an ADSCs injection (treatment group, n=100 knees) in one knee and a saline injection (control group, n=100 knees) in the other knee. The side of the treatment is randomly assigned. Patients are prospectively followed at 1, 3, 6, 12, and 24 months assessing WOMAC, IKDC subjective and objective, EQ-VAS, and Tegner scores. MRI is performed at 12 months, and X-rays are performed with James-Duo assistance at 24 months. Up-to-date 26 patients (26 knees for each groups) were evaluated up to 6 months of follow-up and were included in the current analysis.

Results

No groups reported adverse events. In both groups two patients failed 3 months after treatment, requiring new a injective treatment. At the 6 months follow-up, a significant improvement of all clinical scores was found compared to the baseline evaluation in both groups. The WOMAC score in ADSCs and placebo groups improved from 44.3±14.0 and 45.4±13.9 to 31.9±18.2 and 27.9±20.1, (both p<0.0005) respectively. Similar trends were found for IKDC and VAS scores. No differences were observed between the two groups in terms of absolute values and improvement of the clinical scores.

Conclusion

The preliminary results of this study suggest that ADSCs did not demonstrate a clear superiority at short-term compared to placebo, reporting overall comparable results in terms of clinical scores and failures in patients with knee OA.