Adapting to the acidic environment of the NP: RADA16-PLGA (TGF-ß3) induces chondrogenic differentiation of BMSCs.

Aim: RADA16-PLGA composite scaffolds constructed with simultaneous loading of BMSCs and TGF-ß3 and explored their ability for chondrogenic differentiation in vitro.Methods: The performance of the composite scaffolds is assessed by rheometer assay, electron microscopic structural observation and ELISA release assay. The biosafety of the composite scaffolds is assessed by cytocompatibility assay and cell migration ability. The chondrogenic differentiation ability of composite scaffolds is evaluated by Alisin blue staining, PCR and immunofluorescence staining.Results: The composite scaffold has a good ECM-like structure, the ability to control the release of TGF-ß3 and good biocompatibility. More importantly, the composite scaffolds can induce the differentiation of BMSCs to chondrocytes.Conclusion: Composite scaffolds are expected to enhance the endogenous NP repair process.

[Box: see text].

Matrix Viscoelasticity Tunes the Mechanobiological Behavior of Chondrocytes.

In articular cartilage, the pericellular matrix acting as a specialized mechanical microenvironment modulates environmental signals to chondrocytes through mechanotransduction. Matrix viscoelastic alterations during cartilage development and osteoarthritis (OA) degeneration play an important role in regulating chondrocyte fate and cartilage matrix homeostasis. In recent years, scientists are gradually realizing the importance of matrix viscoelasticity in regulating chondrocyte function and phenotype. Notably, this is an emerging field, and this review summarizes the existing literatures to the best of our knowledge. This review provides an overview of the viscoelastic properties of hydrogels and the role of matrix viscoelasticity in directing chondrocyte behavior. In this review, we elaborated the mechanotransuction mechanisms by which cells sense and respond to the viscoelastic environment and also discussed the underlying signaling pathways. Moreover, emerging insights into the role of matrix viscoelasticity in regulating chondrocyte function and cartilage formation shed light into designing cell-instructive biomaterial. We also describe the potential use of viscoelastic biomaterials in cartilage tissue engineering and regenerative medicine. Future perspectives on mechanobiological comprehension of the viscoelastic behaviors involved in tissue homeostasis, cellular responses, and biomaterial design are highlighted. Finally, this review also highlights recent strategies utilizing viscoelastic hydrogels for designing cartilage-on-a-chip.

Metabolic modulation to improve MSC expansion and therapeutic potential for articular cartilage repair.

BACKGROUND: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. METHODS: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T2) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. RESULTS: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. CONCLUSIONS: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion.

Chemically programmed metabolism drives a superior cell fitness for cartilage regeneration.

The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

Itm2a expression marks periosteal skeletal stem cells that contribute to bone fracture healing.

The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

α7-nAChR/P300/NLRP3-regulated pyroptosis mediated poor articular cartilage quality induced by prenatal nicotine exposure in female offspring rats.

Nicotine is developmentally toxic. Prenatal nicotine exposure (PNE) affects the development of multiple fetal organs and causes susceptibility to a variety of diseases in offspring. In this study, we aimed to investigate the effect of PNE on cartilage development and osteoarthritis susceptibility in female offspring rats. Wistar rats were orally gavaged with nicotine on days 9-20 of pregnancy. The articular cartilage was obtained at gestational day (GD) 20 and postnatal week (PW) 24, respectively. Further, the effect of nicotine on chondrogenic differentiation was explored by the chondrogenic differentiation model in human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). The PNE group showed significantly shallower Safranin O staining and lower Collagen 2a1 content of articular cartilage in female offspring rats. Further, we found that PNE activated pyroptosis in the articular cartilage at GD20 and PW24. In vitro experiments revealed that nicotine inhibited chondrogenic differentiation and activated pyroptosis. After interfering with nod-like receptors3 (NLRP3) expression by SiRNA, it was found that pyroptosis mediated the chondrogenic differentiation inhibition of WJ-MSCs induced by nicotine. In addition, we found that α7-nAChR antagonist α-BTX reversed nicotine-induced NLRP3 and P300 high expression. And, P300 SiRNA reversed the increase of NLRP3 mRNA expression and histone acetylation level in its promoter region induced by nicotine. In conclusion, PNE caused chondrodysplasia and poor articular cartilage quality in female offspring rats. PNE increased the histone acetylation level of NLRP3 promoter region by α7-nAChR/P300, which resulting in the high expression of NLRP3. Further, NLRP3 mediated the inhibition of chondrogenic differentiation by activating pyroptosis.

Interleukin-4-Loaded Gelatin Methacryloyl Hydrogel Promotes Subcutaneous Chondrogenesis of Engineered Auricular Cartilage in a Rabbit Model.

Tissue engineering technology offers a promising solution for ear reconstruction; however, it faces the challenge of foreign body reaction and neocartilage malformation. This study investigates the impact of interleukin-4 (IL-4), an anti-inflammatory factor, on cartilage regeneration of hydrogel encapsulating autologous auricular chondrocytes in a rabbit subcutaneous environment. Initially, we assessed the influence of IL-4 on chondrocyte proliferation and determined the appropriate concentration using the CCK-8 test in vitro. Subsequently, we loaded IL-4 into gelatin methacryloyl (GelMA) hydrogel containing chondrocytes and measured its release profile through ELISA. The constructs were then implanted autologously into rabbits' subcutis, and after 3, 7, 14, and 28 days, cartilage matrix formation was evaluated by histological examinations, and gene expression levels were detected by qRT-PCR. Results demonstrated that IL-4 promotes chondrocyte proliferation in vitro, and maximum release from constructs occurred during the first week. In the rabbit subcutaneous implantation model, IL-4-loaded constructs (20 ng/mL) maintained a superior chondrocytic phenotype compared to controls with increased expression of anti-inflammatory factors. These findings highlight IL-4 as a potential strategy for promoting chondrogenesis in a subcutaneous environment and improving ear reconstruction.

Development of novel osteochondral scaffolds and relatedin vitroenvironment with the aid of chemical engineering principles.

In tissue engineering, collaboration among experts from different fields is needed to design appropriate cell scaffolds and the required three-dimensional environment. Osteochondral tissue engineering is particularly challenging due to the need to provide scaffolds that imitate structural and compositional differences between two neighboring tissues, articular cartilage and bone, and the required complex biophysical environments for cultivating such scaffolds. This work focuses on two key objectives: first, to develop bilayered osteochondral scaffolds based on gellan gum and bioactive glass and, second, to create a biomimetic environment for scaffold characterization by designing and utilizing novel dual-medium cultivation bioreactor chambers. Basic chemical engineering principles were utilized to help achieve both aims. First, a simple heat transport model based on one-dimensional conduction was applied as a guideline for bilayer scaffold preparation, leading to the formation of a gelatinous upper part and a macroporous lower part with a thin, well-integrated interfacial zone. Second, a novel cultivation chamber was developed to be used in a dynamic compression bioreactor to provide possibilities for flow of two different media, such as chondrogenic and osteogenic. These chambers were utilized for characterization of the novel scaffolds with regard to bioactivity and stability under dynamic compression and fluid perfusion over 14 d, while flow distribution under different conditions was analyzed by a tracer method and residence time distribution analysis.

An injectable self-healing alginate hydrogel with desirable mechanical and degradation properties for enhancing osteochondral regeneration.

Articular cartilage and subchondral bone defects have always been problematic because the osteochondral tissue plays a crucial role in the movement of the body and does not recover spontaneously. Here, an injectable hydrogel composed of oxidized sodium alginate/gelatin/chondroitin sulfate (OSAGC) was designed for the minimally invasive treatment and promotion of osteochondral regeneration. The OSAGC hydrogel had a double network based on dynamic covalent bonds, demonstrating commendable injectability and self-healing properties. Chondroitin sulfate was organically bound to the hydrogel network, retaining its own activity and gradually releasing during the degradation process as well as improving mechanical properties. The compressive strength could be increased up to 3 MPa by regulating the concentration of chondroitin sulphate and the oxidation level, and this mechanical stimulation could help repair injured tissue. The OSAGC hydrogel had a favourable affinity to articular cartilage and was able to release active ingredients in a sustained manner over 3 months. The OSAGC showed no cytotoxic effects. Results from animal studies demonstrated its capacity to regenerate new bone tissue in four weeks and new cartilage tissue in twelve weeks. The OSAGC hydrogel represented a promising approach to simplify bone surgery and repair damaged osteochondral tissue.

The cell membrane as biofunctional material for accelerated bone repair.

The cell (plasma) membrane is enriched with numerous receptors, ligands, enzymes, and phospholipids that play important roles in cell-cell and cell-extracellular matrix interactions governing, for instance, tissue development and repair. We previously showed that plasma membrane nanofragments (PMNFs) act as nucleation sites for bone formation in vivo, and induce in vitro mineralization within 1 day. In this study, we optimized the methods for generating, isolating, and applying PMNFs as a cell-free therapeutic to expedite bone defect repair. The PMNFs were isolated from different mouse cell lines (chondrocytes, osteoblasts, and fibroblasts), pre-conditioned, lyophilized, and subsequently transplanted into 2 mm critical-sized calvarial defects in mice (n = 75). The PMNFs from chondrocytes, following a 3-day pre-incubation, significantly accelerated bone repair within 2 weeks, through a coordinated attraction of macrophages, endothelial cells, and osteoblasts to the healing site. In vitro experiments confirmed that PMNFs enhanced cell adhesion. Comparison of the PMNF efficacy with phosphatidylserine, amorphous calcium phosphate (ACP), and living cells confirmed the unique ability of PMNFs to promote accelerated bone repair. Importantly, PMNFs promoted nearly complete integration of the regenerated bone with native tissue after 6 weeks (% non-integrated bone area = 15.02), contrasting with the partial integration (% non-integrated bone area = 56.10; p < 0.01, Student's test) with transplantation of ACP. Vickers microhardness tests demonstrated that the regenerated bone after 6 weeks (30.10 ± 1.75) exhibited hardness similar to native bone (31.07 ± 2.46). In conclusion, this is the first study to demonstrate that cell membrane can be a promising cell-free material with multifaceted biofunctional properties that promote accelerated bone repair. STATEMENT OF SIGNIFICANCE.

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization.

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel's cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel's cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Investigating the differential therapeutic efficacy and mechanisms of human umbilical cord mesenchymal stem cells at various passages in osteoarthritis treatment.

This study aimed to assess the clinical efficacy of umbilical cord mesenchymal stem cells (hUC-MSCs) from different passages (P3, P8, and P13) in the treatment of knee osteoarthritis (OA) and explore the underlying mechanisms. The hUC-MSCs from each passage were characterized and evaluated for their stemness, migration, proliferation, and marker expression. Rats with OA were treated with hUC-MSCs from each passage, and the therapeutic effects were assessed based on knee swelling, discomfort, and pathological examination of the knee joint. Co-culture experiments were conducted to examine the ability of hUC-MSCs to stimulate type II collagen synthesis and inhibit MMP13 expression in chondrocytes. Telomere length and telomerase activity of hUC-MSCs from each passage were measured to investigate the reasons for the observed differences in clinical efficacy. The results revealed that P3 and P8 hUC-MSCs exhibited superior osteogenic and chondrogenic differentiation potential compared to P13, while P13 demonstrated stronger adipogenic differentiation. The wound healing rate was significantly higher in the P3 and P8 groups compared to P13. All hUC-MSC groups expressed high levels of CD90 and CD105, indicating their mesenchymal stem cell characteristics, while CD31 and CD45 were not expressed. CD105 expression was significantly reduced in the P13 group. In the treatment of rat osteoarthritis, there were no significant differences in knee swelling, discomfort, Mankin scores, and pathological findings between P3 and P8 hUC-MSC treatments. However, there was a significant difference between the 8th and 13th passages. Co-culture experiments showed that hUC-MSCs from P3 and P8 enhanced type II collagen synthesis and reduced MMP13 expression in chondrocytes. Although no significant difference was observed between the P3 and P8 groups, a significant difference was found between the P13 and P8 groups. Telomere length analysis revealed that P13 samples had significantly shorter telomeres compared to both P3 and P8. The telomerase activity was positive in P3 and P8 hUC-MSCs, indicating no significant difference between these passages, while it was negative in P13 hUC-MSCs. In conclusion, P3 and P8 hUC-MSCs exhibited superior therapeutic potential for knee osteoarthritis compared to P13, possibly due to their enhanced differentiation capacity and telomerase activity.

Comparative Study of Autogenic and Allogenic Chondrocyte Transplants on Polyethersulfone Scaffolds for Cartilage Regeneration.

The aim of this study was to evaluate the chondrogenic potential of chondrocyte transplants cultured in vitro on polyethersulfone (PES) membranes. Forty-eight rabbits (96 knee joints) were used in the project. The synthetic, macro-porous PES membranes were used as scaffolds. Fragments of articular cartilage were harvested from non-weight-bearing areas of the joints of the animals. Chondrocytes were isolated and then cultivated on PES scaffolds for 3 weeks. The animals were divided into four groups. All the lesions in the articular cartilage were full thickness defects. In Group I, autogenic chondrocytes on PES membranes were transplanted into the defect area; in Group II, allogenic chondrocytes on PES membranes were transplanted into the defect area; in Group III, pure PES membranes were transplanted into the defect area; and in Group IV, lesions were left untreated. Half of the animals from each group were terminated after 8 weeks, and the remaining half were terminated 12 weeks postoperatively. The samples underwent macroscopic evaluation using the Brittberg scale and microscopic evaluation using the O'Driscoll scale. The best regeneration was observed in Groups II and I. In Group I, the results were achieved with two surgeries, while in Group II, only one operation was needed. This indicates that allogenic chondrocytes do not require two surgeries, highlighting the importance of further in vivo studies to better understand this advantage. The success of the study and the desired properties of PES scaffolds are attributed mainly to the presence of sulfonic groups in the structure of the material. These groups, similar to chondroitin sulfate, which naturally occurs in hyaline cartilage, likely enable mutual affinity between the scaffold and cells and promote scaffold colonization by the cells.

Supramolecular Motion Enables Chondrogenic Bioactivity of a Cyclic Peptide Mimetic of Transforming Growth Factor-ß1.

Transforming growth factor (TGF)-ß1 is a multifunctional protein that is essential in many cellular processes that include fibrosis, inflammation, chondrogenesis, and cartilage repair. In particular, cartilage repair is important to avoid physical disability since this tissue does not have the inherent capacity to regenerate beyond full development. We report here on supramolecular coassemblies of two peptide amphiphile molecules, one containing a TGF-ß1 mimetic peptide, and another which is one of two constitutional isomers lacking bioactivity. Using human articular chondrocytes, we investigated the bioactivity of the supramolecular copolymers of each isomer displaying either the previously reported linear form of the mimetic peptide or a novel cyclic analogue. Based on fluorescence depolarization and 1H NMR spin-lattice relaxation times, we found that coassemblies containing the cyclic compound and the most dynamic isomer exhibited the highest intracellular TGF-ß1 signaling and gene expression of cartilage extracellular matrix components. We conclude that control of supramolecular motion is emerging as an important factor in the binding of synthetic molecules to receptors that can be tuned through chemical structure.

Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Sequential release of transforming growth factor ß1 and fibroblast growth factor 2 from nanofibrous scaffolds induces cartilage differentiation of mouse adipose-derived stem cells.

Once damaged, cartilage has poor intrinsic capacity to repair itself. Current cartilage repair strategies cannot restore the damaged tissue sufficiently. It is hypothesized that biomimetic scaffolds, which can recapitulate important properties of the cartilage extracellular matrix, play a beneficial role in supporting cell behaviors such as growth, cartilage differentiation, and integration with native cartilage, ultimately facilitating tissue recovery. Adipose-derived stem cells regenerated cartilage upon the sequential release of transforming growth factor ß1(TGFß1) and fibroblast growth factor 2(FGF2) using a nanofibrous scaffold, in order to get the recovery of functional cartilage. Experiments in vitro have demonstrated that the release sequence of growth factors FGF2 to TGFß1 is the most essential to promote adipose-derived stem cells into chondrocytes that then synthesize collagen II. Mouse subcutaneous implantation indicated that the treatment sequence of FGF2 to TGFß1 was able to significantly induce multiple increase in cartilage regeneration in vivo. This result demonstrates that the group treated with FGF2 to TGFß1 released from a nanofibrous scaffold provides a good strategy for cartilage regeneration by making a favorable microenvironment for cell growth and cartilage regeneration.

Nanoclay Reinforced Integrated Scaffold for Dual-Lineage Regeneration of Cartilage and Subchondral Bone.

Tissue engineering is theoretically considered a promising approach for repairing osteochondral defects. Nevertheless, the insufficient osseous support and integration of the cartilage layer and the subchondral bone frequently lead to the failure of osteochondral repair. Drawing from this, it was proposed that incorporating glycine-modified attapulgite (GATP) into poly(1,8-octanediol-co-citrate) (POC) scaffolds via the one-step chemical cross-linking is proposed to enhance cartilage and subchondral bone defect repair simultaneously. The effects of the GATP incorporation ratio on the physicochemical properties, chondrocyte and MC3T3-E1 behavior, and osteochondral defect repair of the POC scaffold were also evaluated. In vitro studies indicated that the POC/10% GATP scaffold improved cell proliferation and adhesion, maintained cell phenotype, and upregulated chondrogenesis and osteogenesis gene expression. Animal studies suggested that the POC/10% GATP scaffold has significant repair effects on both cartilage and subchondral bone defects. Therefore, the GATP-incorporated scaffold system with dual-lineage bioactivity showed potential application in osteochondral regeneration.

Auricular Chondrocytes as a Cell Source for Scaffold-Free Elastic Cartilage Tissue Engineering.

Current tissue engineering (TE) methods utilize chondrocytes primarily from costal or articular sources. Despite the robust mechanical properties of neocartilages sourced from these cells, the lack of elasticity and invasiveness of cell collection from these sources negatively impact clinical translation. These limitations invited the exploration of naturally elastic auricular cartilage as an alternative cell source. This study aimed to determine if auricular chondrocytes (AuCs) can be used for TE scaffold-free neocartilage constructs and assess their biomechanical properties. Neocartilages were successfully generated from a small quantity of primary neonatal AuCs of three minipig donors (n = 3). Neocartilage constructs had instantaneous moduli of 200.5 kPa ± 43.34 and 471.9 ± 92.8 kPa at 10% and 20% strain, respectively. TE constructs' relaxation moduli (Er) were 36.99 ± 6.47 kPa Er and 110.3 ± 16.99 kPa at 10% and 20% strain, respectively. The Young's modulus was 2.0 MPa ± 0.63, and the ultimate tensile strength was 0.619 ± 0.177 MPa. AuC-derived neocartilages contained 0.144 ± 0.011 µg collagen, 0.185 µg ± 0.002 glycosaminoglycans per µg dry weight, and 1.7e-3 µg elastin per µg dry weight. In conclusion, this study shows that AuCs can be used as a reliable and easily accessible cell source for TE of biomimetic and mechanically robust elastic neocartilage implants.