Extremely low frequency-electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program.

Progressive cartilage deterioration leads to chronic inflammation and loss of joint function, causing osteoarthritis (OA) and joint disease. Although symptoms vary among individuals, the disease can cause severe pain and permanent disability, and effective therapies are urgently needed. Human Adipose-Derived Stem Cells (ADSCs) may differentiate into chondrocytes and are promising for treating OA. Moreover, recent studies indicate that electromagnetic fields (EMFs) could positively affect the chondrogenic differentiation potential of ADSCs. In this work, we investigated the impact of EMFs with frequencies of 35 Hertz and 58 Hertz, referred to as extremely low frequency-EMFs (ELF-EMFs), on the chondrogenesis of ADSCs, cultured in both monolayer and 3D cell micromasses. ADSC cultures were daily stimulated for 36 min with ELF-EMFs or left unstimulated, and the progression of the differentiation process was evaluated by morphological analysis, extracellular matrix deposition, and gene expression profiling of chondrogenic markers. In both culturing conditions, stimulation with ELF-EMFs did not compromise cell viability but accelerated chondrogenesis by enhancing the secretion and deposition of extracellular matrix components at earlier time points in comparison to unstimulated cells. This study showed that, in an appropriate chondrogenic microenvironment, ELF-EMFs enhance chondrogenic differentiation and may be an important tool for supporting and accelerating the treatment of OA through autologous adipose stem cell therapy.

Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-ß signaling inductions and alleviates inflammation in three-dimensional models.

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.

BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A.

Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

ß1-Integrin-Mediated Uptake of Chondrocyte Extracellular Vesicles Regulates Chondrocyte Homeostasis.

Osteoarthritis (OA) is the most prevalent age-related degenerative disorder, which severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), and alterations in chondrocyte EVs during OA have previously been reported. Herein, we investigated factors influencing chondrocyte EV release and the functional role that these EVs exhibit. Both 2D and 3D models of culturing C28I/2 chondrocytes were used for generating chondrocyte EVs. We assessed the effect of these EVs on chondrogenic gene expression as well as their uptake by chondrocytes. Collectively, the data demonstrated that chondrocyte EVs are sequestered within the cartilage ECM and that a bi-directional relationship exists between chondrocyte EV release and changes in chondrogenic differentiation. Finally, we demonstrated that the uptake of chondrocyte EVs is at least partially dependent on ß1-integrin. These results indicate that chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture.

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behaviorin vitro.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Functionally graded hydrogels with opposing biochemical cues for osteochondral tissue engineering.

Osteochondral tissue (OC) repair remains a significant challenge in the field of musculoskeletal tissue engineering. OC tissue displays a gradient structure characterized by variations in both cell types and extracellular matrix components, from cartilage to the subchondral bone. These functional gradients observed in the native tissue have been replicated to engineer OC tissuein vitro. While diverse fabrication methods have been employed to create these microenvironments, emulating the natural gradients and effective regeneration of the tissue continues to present a significant challenge. In this study, we present the design and development of CMC-silk interpenetrating (IPN) hydrogel with opposing dual biochemical gradients similar to native tissue with the aim to regenerate the complete OC unit. The gradients of biochemical cues were generated using an in-house-built extrusion system. Firstly, we fabricated a hydrogel that exhibits a smooth transition of sulfated carboxymethyl cellulose (sCMC) and TGF-ß1 (SCT gradient hydrogel) from the upper to the lower region of the IPN hydrogel to regenerate the cartilage layer. Secondly, a hydrogel with a hydroxyapatite (HAp) gradient (HAp gradient hydrogel) from the lower to the upper region was fabricated to facilitate the regeneration of the subchondral bone layer. Subsequently, we developed a dual biochemical gradient hydrogel with a smooth transition of sCMC + TGF-ß1 and HAp gradients in opposing directions, along with a blend of both biochemical cues in the middle. The results showed that the dual biochemical gradient hydrogels with biochemical cues corresponding to the three zones (i.e. cartilage, interface and bone) of the OC tissue led to differentiation of bone-marrow-derived mesenchymal stem cells to zone-specific lineages, thereby demonstrating their efficacy in directing the fate of progenitor cells. In summary, our study provided a simple and innovative method for incorporating gradients of biochemical cues into hydrogels. The gradients of biochemical cues spatially guided the differentiation of stem cells and facilitated tissue growth, which would eventually lead to the regeneration of the entire OC tissue with a smooth transition from cartilage (soft) to bone (hard) tissues. This promising approach is translatable and has the potential to generate numerous biochemical and biophysical gradients for regeneration of other interface tissues, such as tendon-to-muscle and ligament-to-bone.

rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

An injectable active hydrogel based on BMSC-derived extracellular matrix for cartilage regeneration enhancement.

Articular cartilage injury impairs joint function and necessitates orthopedic intervention to restore the structure and function of the cartilage. Extracellular matrix (ECM) scaffolds derived from bone marrow mesenchymal stem cells (BMSCs) can effectively promote cell adhesion, proliferation, and chondrogenesis. However, pre-shaped ECM scaffolds have limited applicability due to their poor fit with the irregular surface of most articular cartilage defects. In this study, we fabricated an injectable active ECM hydrogel from autologous BMSCs-derived ECM by freeze-drying, liquid nitrogen milling, and enzymatic digestion. Moreover, our in vitro and in vivo results demonstrated that the prepared hydrogel enhanced chondrocyte adhesion and proliferation, chondrogenesis, cartilage regeneration, and integration with host tissue, respectively. These findings indicate that active ECM components can provide trophic support for cell proliferation and differentiation, restoring the structure and function of damaged cartilage.

Mid-term results of autologous matrix-induced chondrogenesis for large chondral defects in hips with femoroacetabular impingement syndrome.

Aims: The purpose of this study was to evaluate the mid-term outcomes of autologous matrix-induced chondrogenesis (AMIC) for the treatment of larger cartilage lesions and deformity correction in hips suffering from symptomatic femoroacetabular impingement (FAI). Methods: This single-centre study focused on a cohort of 24 patients with cam- or pincer-type FAI, full-thickness femoral or acetabular chondral lesions, or osteochondral lesions ≥ 2 cm2, who underwent surgical hip dislocation for FAI correction in combination with AMIC between March 2009 and February 2016. Baseline data were retrospectively obtained from patient files. Mid-term outcomes were prospectively collected at a follow-up in 2020: cartilage repair tissue quality was evaluated by MRI using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. Patient-reported outcome measures (PROMs) included the Oxford Hip Score (OHS) and Core Outcome Measure Index (COMI). Clinical examination included range of motion, impingement tests, and pain. Results: A total of 12 hips from 11 patients were included (ten males, one female, mean age 26.8 years (SD 5.0), mean follow-up 6.2 years (SD 5.2 months)). The mean postoperative MOCART score was 66.3 (SD 16.3). None of the patients required conversion to total hip arthroplasty. Two patients had anterior impingement. External hip rotation was moderately limited in four patients. There was a correlation between MOCART and follow-up time (rs = -0.61; p = 0.035), but not with initial cartilage damage, age, BMI, or imaging time delay before surgery. PROMs improved significantly: OHS from 37.4 to 42.7 (p = 0.014) and COMI from 4.1 to 1.6 (p = 0.025). There was no correlation between MOCART and PROMs. Conclusion: Based on the reported mid-term results, we consider AMIC as an encouraging treatment option for large cartilage lesions of the hip. Nonetheless, the clinical evidence of AMIC in FAI patients remains to be determined, ideally in the context of randomized controlled trials.

"A lactose-modified chitosan accelerates chondrogenic differentiation in mesenchymal stem cells spheroids".

Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.

Regeneration: AT-AMIC Technique: Limits and Indication.

Osteochondral lesion of the talus (OLT) is a commune cause of chronic ankle pain. Symptomatic lesions require surgical treatment. Currently, lesions with diameter less than 107.4 mm2 are treated with bone marrow stimulating technique with notable success rate. However, more extensive lesions show less predictable surgical results. Autologous matrix-induced chondrogenesis has proven to provide satisfactory medium and long-term results on OLTs. In the current review, we describe an all-arthroscopic technique and the Milan-Tel Aviv lesion assessment protocol.

Unlocking Potential: Low Bovine Serum Albumin Enhances the Chondrogenicity of Human Adipose-Derived Stromal Cells in Pellet Cultures.

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

Induced pluripotent stem cells in cartilage tissue engineering: a literature review.

Osteoarthritis (OA) is a long-term, persistent joint disorder characterized by bone and cartilage degradation, resulting in tightness, pain, and restricted movement. Current attempts in cartilage regeneration are cell-based therapies using stem cells. Multipotent stem cells, such as mesenchymal stem cells (MSCs), and pluripotent stem cells, such as embryonic stem cells (ESCs), have been used to regenerate cartilage. However, since the discovery of human-induced pluripotent stem cells (hiPSCs) in 2007, it was seen as a potential source for regenerative chondrogenic therapy as it overcomes the ethical issues surrounding the use of ESCs and the immunological and differentiation limitations of MSCs. This literature review focuses on chondrogenic differentiation and 3D bioprinting technologies using hiPSCS, suggesting them as a viable source for successful tissue engineering. METHODS: A literature search was conducted using scientific search engines, PubMed, MEDLINE, and Google Scholar databases with the terms 'Cartilage tissue engineering' and 'stem cells' to retrieve published literature on chondrogenic differentiation and tissue engineering using MSCs, ESCs, and hiPSCs. RESULTS: hiPSCs may provide an effective and autologous treatment for focal chondral lesions, though further research is needed to explore the potential of such technologies. CONCLUSIONS: This review has provided a comprehensive overview of these technologies and the potential applications for hiPSCs in regenerative medicine.

Reciprocal negative feedback between Prrx1 and miR-140-3p regulates rapid chondrogenesis in the regenerating antler.

During growth phase, antlers exhibit a very rapid rate of chondrogenesis. The antler is formed from its growth center reserve mesenchyme (RM) cells, which have been found to be the derivatives of paired related homeobox 1 (Prrx1)-positive periosteal cells. However, the underlying mechanism that drives rapid chondrogenesis is not known. Herein, the miRNA expression profiles and chromatin states of three tissue layers (RM, precartilage, and cartilage) at different stages of differentiation within the antler growth center were analyzed by RNA-sequencing and ATAC-sequencing. We found that miR-140-3p was the miRNA that exhibited the greatest degree of upregulation in the rapidly growing antler, increasing from the RM to the cartilage layer. We also showed that Prrx1 was a key upstream regulator of miR-140-3p, which firmly confirmed by Prrx1 CUT&Tag sequencing of RM cells. Through multiple approaches (three-dimensional chondrogenic culture and xenogeneic antler model), we demonstrated that Prrx1 and miR-140-3p functioned as reciprocal negative feedback in the antler growth center, and downregulating PRRX1/upregulating miR-140-3p promoted rapid chondrogenesis of RM cells and xenogeneic antler. Thus, we conclude that the reciprocal negative feedback between Prrx1 and miR-140-3p is essential for balancing mesenchymal proliferation and chondrogenic differentiation in the regenerating antler. We further propose that the mechanism underlying chondrogenesis in the regenerating antler would provide a reference for helping understand the regulation of human cartilage regeneration and repair.

Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept.

BACKGROUND: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-ß is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-ß can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-ß receptors during MSC in vitro chondrogenesis. METHODS: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-ß and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. RESULTS: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. CONCLUSIONS: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-ß that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-ß-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

Specific lipid magnetic sphere sorted CD146-positive bone marrow mesenchymal stem cells can better promote articular cartilage damage repair.

BACKGROUND: The characteristics and therapeutic potential of subtypes of bone marrow mesenchymal stem cells (BMSCs) are largely unknown. Also, the application of subpopulations of BMSCs in cartilage regeneration remains poorly characterized. The aim of this study was to explore the regenerative capacity of CD146-positive subpopulations of BMSCs for repairing cartilage defects. METHODS: CD146-positive BMSCs (CD146 + BMSCs) were sorted by self-developed CD146-specific lipid magnetic spheres (CD146-LMS). Cell surface markers, viability, and proliferation were evaluated in vitro. CD146 + BMSCs were subjected to in vitro chondrogenic induction and evaluated for chondrogenic properties by detecting mRNA and protein expression. The role of the CD146 subpopulation of BMSCs in cartilage damage repair was assessed by injecting CD146 + BMSCs complexed with sodium alginate gel in the joints of a mouse cartilage defect model. RESULTS: The prepared CD146-LMS had an average particle size of 193.7 ± 5.24 nm, an average potential of 41.9 ± 6.21 mv, and a saturation magnetization intensity of 27.2 Am2/kg, which showed good stability and low cytotoxicity. The sorted CD146 + BMSCs highly expressed stem cell and pericyte markers with good cellular activity and cellular value-added capacity. Cartilage markers Sox9, Collagen II, and Aggrecan were expressed at both protein and mRNA levels in CD146 + BMSCs cells after chondrogenic induction in vitro. In a mouse cartilage injury model, CD146 + BMSCs showed better function in promoting the repair of articular cartilage injury. CONCLUSION: The prepared CD146-LMS was able to sort out CD146 + BMSCs efficiently, and the sorted subpopulation of CD146 + BMSCs had good chondrogenic differentiation potential, which could efficiently promote the repair of articular cartilage injury, suggesting that the sorted CD146 + BMSCs subpopulation is a promising seed cell for cartilage tissue engineering.