Chondrogenic Factors Research Articles

9267 Targeting IGF-I to Growth Plate Cartilage Augments Therapeutic Effects on Skeletal Growth and Reduces Hypoglycemic Effects in Mice

Abstract Disclosure: K. Tailor: None. T. Stowe: None. J. Ree: None. B. Ventura: None. J. Deursen: None. R. O'Brien: None. J. Baron: None. J. Lui: None. Recombinant human growth hormone (GH) is commonly used to treat short stature in children but has limited efficacy in severe, non-GH-deficient conditions, such as skeletal dysplasias, and has adverse off-target effects. Because short stature is the result of reduced growth plate chondrogenesis, we hypothesized that targeting chondrogenic growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing adverse effects on non-target tissues. Toward this end, we previously developed fusion proteins conjugating insulin-like growth factor-1 (IGF-1) with a single-chain human monoclonal antibody fragment (ScFv) targeting matrillin-3 (1), a protein specifically expressed in cartilage matrix, and demonstrated increased on-target therapeutic efficacy at the growth plate and decreased off-target effects in a GH-deficient (lit) mouse model (2). In the current study, we established a second GH-deficiency model by administration of a GH antagonist, pegvisomant (PEG), in C57BL/6 wild-type male mice. Alternate-day injection of pegvisomant at 40 mg/kg for 7 days reduced body weight, serum IGF-1 levels, and growth plate height in mice. Using this model, we found that once-daily SQ administration of cartilage-targeted IGF-I (named CV1574-1) for five days increased body weight and growth plate height greater than an equimolar dose of IGF-I itself administered twice daily. Hypoglycemia is an adverse effect of recombinant IGF-1 treatment. Therefore, we also monitored blood glucose levels after a single SQ injection of CV1574-1 and found that this fusion protein showed a reduced hypoglycemic effect compared to injection of IGF-I itself. Lastly, to gain mechanistic insight into the role of matrillin-3 targeting on therapeutic efficacy, we assessed the ability of CV1574-1 to induce pAKT signaling in vitro. Transient exposure to CV1574-1 caused more prolonged induction of pAKT signal in wild-type rat chondrosarcoma (RCS) cells compared to matrillin-3-knockout RCS cells, suggesting that matrillin-3 targeting extends the duration of IGF-1 receptor activation. To summarize, the current findings demonstrate that CV1574-1, a fusion protein that targets IGF-1 to cartilage, improves efficacy at the growth plate, reduces hypoglycemic effects, and, in vitro, prolongs receptor activation. Our study provides further evidence that cartilage-targeted therapy has the potential to provide new pharmacological approaches for the treatment of childhood linear growth disorders, including skeletal dysplasias and growth failure due to systemic disease. Reference:(1) Cheung et al. Pharma Res 2015, 32(7):2439-49.(2) Lui et al. Molecular Therapy 2019, 27(3):673-680 Presentation: 6/3/2024

7237 Targeting IGF-I to Growth Plate Cartilage Augments Therapeutic Effects on Skeletal Growth and Reduces Hypoglycemic Effects in Mice

Abstract Disclosure: K. Tailor: None. T. Stowe: None. J. Ree: None. B. Ventura: None. J. Deursen: None. R. O'Brien: None. J. Baron: None. J. Lui: None. Recombinant human growth hormone (GH) is commonly used to treat short stature in children but has limited efficacy in severe, non-GH-deficient conditions, such as skeletal dysplasias, and has adverse off-target effects. Because short stature is the result of reduced growth plate chondrogenesis, we hypothesized that targeting chondrogenic growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing adverse effects on non-target tissues. Toward this end, we previously developed fusion proteins conjugating insulin-like growth factor-1 (IGF-1) with a single-chain human monoclonal antibody fragment (ScFv) targeting matrillin-3 (1), a protein specifically expressed in cartilage matrix, and demonstrated increased on-target therapeutic efficacy at the growth plate and decreased off-target effects in a GH-deficient (lit) mouse model (2). In the current study, we established a second GH-deficiency model by administration of a GH antagonist, pegvisomant (PEG), in C57BL/6 wild-type male mice. Alternate-day injection of pegvisomant at 40 mg/kg for 7 days reduced body weight, serum IGF-1 levels, and growth plate height in mice. Using this model, we found that once-daily SQ administration of cartilage-targeted IGF-I (named CV1574-1) for five days increased body weight and growth plate height greater than an equimolar dose of IGF-I itself administered twice daily. Hypoglycemia is an adverse effect of recombinant IGF-1 treatment. Therefore, we also monitored blood glucose levels after a single SQ injection of CV1574-1 and found that this fusion protein showed a reduced hypoglycemic effect compared to injection of IGF-I itself. Lastly, to gain mechanistic insight into the role of matrillin-3 targeting on therapeutic efficacy, we assessed the ability of CV1574-1 to induce pAKT signaling in vitro. Transient exposure to CV1574-1 caused more prolonged induction of pAKT signal in wild-type rat chondrosarcoma (RCS) cells compared to matrillin-3-knockout RCS cells, suggesting that matrillin-3 targeting extends the duration of IGF-1 receptor activation. To summarize, the current findings demonstrate that CV1574-1, a fusion protein that targets IGF-1 to cartilage, improves efficacy at the growth plate, reduces hypoglycemic effects, and, in vitro, prolongs receptor activation. Our study provides further evidence that cartilage-targeted therapy has the potential to provide new pharmacological approaches for the treatment of childhood linear growth disorders, including skeletal dysplasias and growth failure due to systemic disease. Reference:(1) Cheung et al. Pharma Res 2015, 32(7):2439-49.(2) Lui et al. Molecular Therapy 2019, 27(3):673-680 Presentation: 6/3/2024

Macroporous PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates Prepared via Radical-Free Cross-Linking for Cartilage Tissue Engineering.

Trauma or repeated damage to joints can result in focal cartilage defects, significantly elevating the risk of osteoarthritis. Damaged cartilage has an inherently limited self-healing capacity and remains an urgent unmet clinical need. Consequently, there is growing interest in biodegradable hydrogels as potential scaffolds for the repair or reconstruction of cartilage defects. Here, we developed a biodegradable and macroporous hybrid double-network (DN) cryogel by combining two independently cross-linked networks of multiarm polyethylene glycol (PEG) acrylate and alginate.Hybrid DN cryogels are formed using highly biocompatible click reactions for the PEG network and ionic bonding for the alginate network. By judicious selection of various structurally similar cross-linkers to form the PEG network, we can generate hybrid DN cryogels with customizable degradation kinetics. The resulting PEG-alginate hybrid DN cryogels have an interconnected macroporous structure, high mechanical strength, and rapid swelling kinetics. The interconnected macropores in the cryogels support efficient mesenchymal stem cell infiltration at a high density. Finally, we demonstrate that PEG-alginate hybrid DN cryogels allow sustained release of chondrogenic growth factors and support chondrogenic differentiation of mouse mesenchymal stem cells. This study provides a novel method to generate macroporous hybrid DN cryogels with customizable degradation rates and a potential scaffold for cartilage tissue engineering.

A Novel Scaffold of Icariin/Porous Magnesium Alloy-Repaired Knee Cartilage Defect in Rat by Wnt/β-Catenin Signaling Pathway.

Cartilage defects caused by joint diseases are difficult to treat clinically. Tissue engineering materials provide a new means to promote the repair of cartilage defects. The purpose of this study is to design a novel scaffold of porous magnesium alloy loaded with icariin and sustained release in order to explore the effect and possible mechanism of this scaffold in repairing SD rat knee articular cartilage defect. We constructed a novel type of icariin/porous magnesium alloy scaffold, observed the structure of the scaffold by electron microscope, detected the drug release of icariin in the scaffold and the biological safety, and established an animal model of cartilage defect in the femoral intercondylar fossa of the knee joint in rats; the scaffold was placed in the defect. After 12 weeks of repair, the rat knee articular cartilage repair was evaluated by gross specimens and micro-CT, HE, safranin O-fast green, and toluidine blue staining combined with the modified Mankin's score. The protein expressions of the Wnt/β-catenin signaling pathway-related factors (β-catenin, Wnt5a, Wnt1, sFRP1) and chondrogenic differentiation-related factors (Sox9, Aggrecan, Col2α1) were detected by immunohistochemical staining. We found that the novel scaffold of icariin/porous magnesium alloy can release icariin slowly and has biosafety in rats. Compared with other groups, icariin/porous magnesium alloy can significantly promote the repair of cartilage defects and the expressions of β-catenin, Wnt5a, Wnt1, Sox9, Aggrecan, and Col2α1 (P < 0.05). This novel scaffold can promote the repair of rat knee cartilage defects, and this process may be achieved by activating the Wnt/β-catenin signaling pathway.

Biomimetic kartogenin containing κ-carrageenan hydrogel for nucleus pulposus regeneration

Intervertebral disc degeneration is a clinical disease that reduces the quality of patient's life. The degeneration usually initiates in the nucleus pulposus (NP), hence the use of hydrogels represents a promising therapeutic approach. However, the viscoelastic nature of hydrogel and its ability to provide biomimetic architecture and biochemical cues influence the regeneration capability. This study focused on tuning the physical nature of a glycosaminoglycan hydrogel (κ-carrageenan) as well as the release kinetics of a chondrogenic factor (kartogenin - KGN) through physical cross-linking. For this, κ-carrageenan was cross linked with 2.5 % and 5 % potassium chloride (KCl) for 15 and 30 min and loaded with KGN molecule at 50 μM and 100 μM. The tight network structure with low water retention and degradation property was seen in hydrogel cross-linked with increased KCl concentration and time. However, optimal degradation along with NP mimicking viscoelastic nature was exhibited by 5 wt% KCl treated hydrogel (H3 hydrogel). All hydrogel groups exhibited burst KGN release at 24 h followed by a sustained release for 5 days. However, hydrogel cross-linked with 5 wt% KCl enhanced chondrogenic differentiation, mainly at lower KGN dose. In summary, this study shows the potential application of biomimetic KGN laden carrageenan hydrogel in NP regeneration.

Cartilage lacuna-biomimetic hydrogel microspheres endowed with integrated biological signal boost endogenous articular cartilage regeneration

Despite numerous studies on chondrogenesis, the repair of cartilage—particularly the reconstruction of cartilage lacunae through an all-in-one advanced drug delivery system remains limited. In this study, we developed a cartilage lacuna-like hydrogel microsphere system endowed with integrated biological signals, enabling sequential immunomodulation and endogenous articular cartilage regeneration. We first integrated the chondrogenic growth factor transforming growth factor-β3 (TGF-β3) into mesoporous silica nanoparticles (MSNs). Then, TGF-β3@MSNs and insulin-like growth factor 1 (IGF-1) were encapsulated within microspheres made of polydopamine (pDA). In the final step, growth factor-loaded MSN@pDA and a chitosan (CS) hydrogel containing platelet-derived growth factor-BB (PDGF-BB) were blended to produce growth factors loaded composite microspheres (GFs@μS) using microfluidic technology. The presence of pDA reduced the initial acute inflammatory response, and the early, robust release of PDGF-BB aided in attracting endogenous stem cells. Over the subsequent weeks, the continuous release of IGF-1 and TGF-β3 amplified chondrogenesis and matrix formation. μS were incorporated into an acellular cartilage extracellular matrix (ACECM) and combined with a polydopamine-modified polycaprolactone (PCL) structure to produce a tissue-engineered scaffold that mimicked the structure of the cartilage lacunae evenly distributed in the cartilage matrix, resulting in enhanced cartilage repair and patellar cartilage protection. This research provides a strategic pathway for optimizing growth factor delivery and ensuring prolonged microenvironmental remodeling, leading to efficient articular cartilage regeneration.

The role of semaphorin 3A on chondrogenic differentiation

Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.

A Combined Treatment of BMP2 and Soluble VEGFR1 for the Enhancement of Tendon-Bone Healing by Regulating Injury-Activated Skeletal Stem Cell Lineage

Background: Bone morphogenetic protein 2 (BMP2) is an appealing osteogenic and chondrogenic growth factor for promoting tendon-bone healing. Recently, it has been reported that soluble vascular endothelial growth factor (VEGF) receptor 1 (sVEGFR1) (a VEGF receptor antagonist) could enhance BMP2-induced bone repair and cartilage regeneration; thus, their combined application may represent a promising treatment to improve tendon-bone healing. Moreover, BMP2 could stimulate skeletal stem cell (SSC) expansion and formation, which is responsible for wounded tendon-bone interface repair. However, whether the codelivery of BMP2 and sVEGFR1 increases tendon enthesis injury–activated SSCs better than does BMP2 alone needs further research. Purpose: To study the effect of BMP2 combined with sVEGFR1 on tendon-bone healing and injury-activated SSC lineage. Study Design: Controlled laboratory study. Methods: A total of 128 C57BL/6 mice that underwent unilateral supraspinatus tendon detachment and repair were randomly assigned to 4 groups: (1) untreated control group; (2) hydrogel group, which received a local injection of the blank hydrogel at the injured site; (3) BMP2 group, which received an injection of hydrogel with BMP2; and (4) BMP2 with sVEGFR1 group, which received an injection of hydrogel with BMP2 and sVEGFR1. Histology, micro–computed tomography, and biomechanical tests were conducted to evaluate tendon-bone healing at 4 and 8 weeks after surgery. In addition, flow cytometry was performed to detect the proportion of SSCs and their downstream differentiated subtypes, including bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors within supraspinatus tendon enthesis at 1 week postoperatively. Results: The repaired interface in BMP2 with sVEGFR1 group showed a significantly improved collagen fiber continuity, increased fibrocartilage, greater newly formed bone, and elevated mechanical properties compared with the other 3 groups. There were more SSCs; bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors in the BMP2 with sVEGFR1 group than that in the other groups. Conclusion: Our study suggests that the combined delivery of BMP2 and sVEGFR1 could promote tendon-bone healing and stimulate the expansion of SSCs and their downstream progeny within the injured tendon-bone interface. Clinical Relevance: Combining BMP2 with sVEGFR1 may be a good clinical treatment for wounded tendon enthesis healing.

Skeletal growth is enhanced by a shared role for SOX8 and SOX9 in promoting reserve chondrocyte commitment to columnar proliferation

SOX8 was linked in a genome-wide association study to human height heritability, but roles in chondrocytes for this close relative of the master chondrogenic transcription factor SOX9 remain unknown. We undertook here to fill this knowledge gap. High-throughput assays demonstrate expression of human SOX8 and mouse Sox8 in growth plate cartilage. In situ assays show that Sox8 is expressed at a similar level as Sox9 in reserve and early columnar chondrocytes and turned off when Sox9 expression peaks in late columnar and prehypertrophic chondrocytes. Sox8-/- mice and Sox8fl/flPrx1Cre and Sox9fl/+Prx1Cre mice (inactivation in limb skeletal cells) have a normal or near normal skeletal size. In contrast, juvenile and adult Sox8fl/flSox9fl/+Prx1Cre compound mutants exhibit a 15 to 20% shortening of long bones. Their growth plate reserve chondrocytes progress slowly toward the columnar stage, as witnessed by a delay in down-regulating Pthlh expression, in packing in columns and in elevating their proliferation rate. SOX8 or SOX9 overexpression in chondrocytes reveals not only that SOX8 can promote growth plate cell proliferation and differentiation, even upon inactivation of endogenous Sox9, but also that it is more efficient than SOX9, possibly due to greater protein stability. Altogether, these findings uncover a major role for SOX8 and SOX9 in promoting skeletal growth by stimulating commitment of growth plate reserve chondrocytes to actively proliferating columnar cells. Further, by showing that SOX8 is more chondrogenic than SOX9, they suggest that SOX8 could be preferred over SOX9 in therapies to promote cartilage formation or regeneration in developmental and degenerative cartilage diseases.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Rat Knee Osteoarthritis via Activating Wnt/ β-catenin Signaling Pathway.

Osteoarthritis (OA) is a chronic disease characterized by joint cartilage degeneration, destruction, and osteogenic hyperplasia. Human umbilical cord mesenchymal stem cells (hUCMSCs) have attracted increasing research interest due to their high clonogenic, proliferative, and migratory potential, as well as their improved secretion of relevant chondrogenic factors. This study evaluated the therapeutic potential and underlying mechanism of hUC-MSCs in alleviating pathological symptoms of OA. For the in vivo study, OA rats were established by the Hulth method to observe the therapeutic effect of intra-articular injection of hUC-MSCs. X-ray tests, gross observations, and histological and immunohistochemical assessments were conducted in rats. Levels of interleukin-1 beta (IL-1β), IL-6, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor matrix metalloproteinase-1 in rats' synovial fluid were measured using enzyme-linked immunosorbent assay kits. For the in vitro study, hUC-MSCs and chondrocytes were cultured to explore the effect and underlying mechanisms of hUC-MSCs on OA. Apoptosis, proliferation, and glycosaminoglycan (GAG) were measured in the chondrocytes. The relative expression of aggrecan, COL-2, and SOX-9 mRNA was quantified by real-time polymerase chain reaction. Expressions of Wnt/β-catenin signaling molecules were measured by Western blot. We found that intra-articular injection of hUC-MSCs reduced the combined score, increased the expression of collagen II, and decreased the expression of MMP-13, IL-1β, and IL-6 in rat knee joints. Additionally, hUC-MSCs increased the content of GAGs, inhibited chondrocyte apoptosis, and promoted chondrocyte proliferation. The expression of aggrecan, COL-2, and SOX-9 mRNA in chondrocytes was promoted by hUC-MSCs via activation of the Wnt/β-catenin signaling pathway. Overall, this study demonstrated that hUC-MSCs induce the secretion of some cytokines via the paracrine function to activate the Wnt/β-catenin signaling pathway to reduce the pathological condition of OA and maintain the proper expression of cytokines and extracellular matrix proteins.

Temporal control in shell-core structured nanofilm for tracheal cartilage regeneration: synergistic optimization of anti-inflammation and chondrogenesis.

Cartilage tissue engineering offers hope for tracheal cartilage defect repair. Establishing an anti-inflammatory microenvironment stands as a prerequisite for successful tracheal cartilage restoration, especially in immunocompetent animals. Hence, scaffolds inducing an anti-inflammatory response before chondrogenesis are crucial for effectively addressing tracheal cartilage defects. Herein, we develop a shell-core structured PLGA@ICA-GT@KGN nanofilm using poly(lactic-co-glycolic acid) (PLGA) and icariin (ICA, an anti-inflammatory drug) as the shell layer and gelatin (GT) and kartogenin (KGN, a chondrogenic factor) as the core via coaxial electrospinning technology. The resultant PLGA@ICA-GT@KGN nanofilm exhibited a characteristic fibrous structure and demonstrated high biocompatibility. Notably, it showcased sustained release characteristics, releasing ICA within the initial 0 to 15 days and gradually releasing KGN between 11 and 29 days. Subsequent in vitro analysis revealed the potent anti-inflammatory capabilities of the released ICA from the shell layer, while the KGN released from the core layer effectively induced chondrogenic differentiation of bone marrow stem cells (BMSCs). Following this, the synthesized PLGA@ICA-GT@KGN nanofilms were loaded with BMSCs and stacked layer by layer, adhering to a 'sandwich model' to form a composite sandwich construct. This construct was then utilized to repair circular tracheal defects in a rabbit model. The sequential release of ICA and KGN facilitated by the PLGA@ICA-GT@KGN nanofilm established an anti-inflammatory microenvironment before initiating chondrogenic induction, leading to effective tracheal cartilage restoration. This study underscores the significance of shell-core structured nanofilms in temporally regulating anti-inflammation and chondrogenesis. This approach offers a novel perspective for addressing tracheal cartilage defects, potentially revolutionizing their treatment methodologies.

Resveratrol's Impact on the Chondrogenic Reagents' Effects in Cell Sheet Cultures of Wharton's Jelly-Derived MSCs.

Human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation.

Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis.

The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

BMP9 is a potent inducer of chondrogenesis, volumetric expansion and collagen type II accumulation in bovine auricular cartilage chondroprogenitors.

Reconstruction of the outer ear currently requires harvesting of cartilage from the posterior of the auricle or ribs leading to pain and donor site morbidity. An alternative source for auricular reconstruction is in vitro tissue engineered cartilage using stem/progenitor cells. Several candidate cell-types have been studied with tissue-specific auricular cartilage progenitor cells (AuCPC) of particular interest. Whilst chondrogenic differentiation of competent stem cells using growth factor TGFβ1 produces cartilage this tissue is frequently fibrocartilaginous and lacks the morphological features of hyaline cartilage. Recent work has shown that growth factor BMP9 is a potent chondrogenic and morphogenetic factor for articular cartilage progenitor cells, and we hypothesised that this property extends to cartilage-derived progenitors from other tissues. In this study we show monoclonal populations of AuCPCs from immature and mature bovine cartilage cultured with BMP9 produced cartilage pellets have 3-5-fold greater surface area in sections than those grown with TGFβ1. Increased volumetric growth using BMP9 was due to greater sGAG deposition in immature pellets and significantly greater collagen accumulation in both immature and mature progenitor pellets. Polarised light microscopy and immunohistochemical analyses revealed that the organisation of collagen fibrils within pellets is an important factor in the growth of pellets. Additionally, chondrocytes in BMP9 stimulated cell pellets had larger lacunae and were more evenly dispersed throughout the extracellular matrix. Interestingly, BMP9 tended to normalise the response of immature AuCPC monoclonal cell lines to differentiation cues whereas cells exhibited more variation under TGFβ1. In conclusion, BMP9 appears to be a potent inducer of chondrogenesis and volumetric growth for AuCPCs a property that can be exploited for tissue engineering strategies for reconstructive surgery though with the caveat of negligible elastin production following 21-day treatment with either growth factor.

PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice

BackgroundInduction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE−/−) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE−/− mouse model of atherosclerosis and under the influence of a high-fat diet.MethodsFor this purpose, PACAP−/−/ApoE−/−, PAC1−/−/ApoE−/−, and ApoE−/− mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified.ResultsApoE−/− mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1−/−/ApoE−/− mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE−/− mice. In contrast, PACAP deficiency in ApoE−/− mice did not affect the chondrogenic signature under either diet.ConclusionsOur data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell–based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro–computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

Administration of mRNA-Nanomedicine-Augmented Calvarial Defect Healing via Endochondral Ossification.

Large-area craniofacial defects remain a challenge for orthopaedists, hastening the need to develop a facile and safe tissue engineering strategy; osteoconductive material and a combination of optimal growth factors and microenvironment should be considered. Faced with the unmet need, we propose that abundant cytokines and chemokines can be secreted from the bone defect, provoking the infiltration of endogenous stem cells to assist bone regeneration. We can provide a potent mRNA medicine cocktail to promptly initiate the formation of bone templates, osteogenesis, and subsequent bone matrix deposition via endochondral ossification, which may retard rapid fibroblast infiltration and prevent the formation of atrophic non-union. We explored the mutual interaction of BMP2 and TGFβ3 mRNA, both potent chondrogenic factors, on inducing endochondral ossification; examined the influence of in vitro the transcribed polyA tail length on mRNA stability; prepared mRNA nanomedicine using a PEGylated polyaspartamide block copolymer loaded in a gelatin sponge and grafted in a critical-sized calvarial defect; and evaluated bone regeneration using histological and μCT examination. The BMP2 and TGFβ3 composite mRNA nanomedicine resulted in over 10-fold new bone volume (BV) regeneration in 8 weeks than the BMP2 mRNA nanomedicine administration alone, demonstrating that the TGFβ3 mRNA nanomedicine synergistically enhances the bone's formation capability, which is induced by BMP2 mRNA nanomedicine. Our data demonstrated that mRNA-medicine-mediated endochondral ossification provides an alternative cell-free tissue engineering methodology for guiding craniofacial defect healing.

Hypoxia Preconditioned Serum (HPS) Promotes Proliferation and Chondrogenic Phenotype of Chondrocytes In Vitro.

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects remains challenging in terms of maintaining chondrogenic phenotype during in vitro chondrocyte expansion. Growth factor supplementation has been found supportive in improving ACI outcomes by promoting chondrocyte redifferentiation. Here, we analysed the chondrogenic growth factor concentrations in the human blood-derived secretome of Hypoxia Preconditioned Serum (HPS) and assessed the effect of HPS-10% and HPS-40% on human articular chondrocytes from osteoarthritic cartilage at different time points compared to normal fresh serum (NS-10% and NS-40%) and FCS-10% culture conditions. In HPS, the concentrations of TGF-beta1, IGF-1, bFGF, PDGF-BB and G-CSF were found to be higher than in NS. Chondrocyte proliferation was promoted with higher doses of HPS (HPS-40% vs. HPS-10%) and longer stimulation (4 vs. 2 days) compared to FCS-10%. On day 4, immunostaining of the HPS-10%-treated chondrocytes showed increased levels of collagen type II compared to the other conditions. The promotion of the chondrogenic phenotype was validated with quantitative real-time PCR for the expression of collagen type II (COL2A1), collagen type I (COL1A1), SOX9 and matrix metalloproteinase 13 (MMP13). We demonstrated the highest differentiation index (COL2A1/COL1A1) in HPS-10%-treated chondrocytes on day 4. In parallel, the expression of differentiation marker SOX9 was elevated on day 4, with HPS-10% higher than NS-10/40% and FCS-10%. The expression of the cartilage remodelling marker MMP13 was comparable across all culture conditions. These findings implicate the potential of HPS-10% to improve conventional FCS-based ACI culture protocols by promoting the proliferation and chondrogenic phenotype of chondrocytes during in vitro expansion.

3D bioprinted hydrogel/polymer scaffold with factor delivery and mechanical support for growth plate injury repair.

Introduction: Growth plate injury is a significant challenge in clinical practice, as it could severely affect the limb development of children, leading to limb deformity. Tissue engineering and 3D bioprinting technology have great potential in the repair and regeneration of injured growth plate, but there are still challenges associated with achieving successful repair outcomes. Methods: In this study, GelMA hydrogel containing PLGA microspheres loaded with chondrogenic factor PTH(1-34) was combined with BMSCs and Polycaprolactone (PCL) to develop the PTH(1-34)@PLGA/BMSCs/GelMA-PCL scaffold using bio-3D printing technology. Results: The scaffold exhibited a three-dimensional interconnected porous network structure, good mechanical properties, biocompatibility, and was suitable for cellchondrogenic differentiation. And a rabbit model of growth plate injury was appliedto validate the effect of scaffold on the repair of injured growth plate. The resultsshowed that the scaffold was more effective than injectable hydrogel in promotingcartilage regeneration and reducing bone bridge formation. Moreover, the addition ofPCL to the scaffold provided good mechanical support, significantly reducing limbdeformities after growth plate injury compared with directly injected hydrogel. Discussion: Accordingly, our study demonstrates the feasibility of using 3D printed scaffolds for treating growth plate injuries and could offer a new strategy for the development of growth plate tissue engineering therapy.