Mature Cartilage Research Articles

Mineralization of equine proximal sesamoid bones precedes articular cartilage and fibrocartilaginous enthesis maturation in early postgestational development.

To describe the development and maturation of equine proximal sesamoid bones (PSBs) in fetuses and young horses using radiography, microcomputed (micro)-CT, and histology. A descriptive study. Forelimb PSBs from 12 equids ranging in age from 105 days of gestation to 540 days postgestation were evaluated. Radiography was used for preliminary assessment of metacarpophalangeal joint and PSB mineralization, and micro-CT imaging was performed to assess mineralized PSBs. Tissue volume, bone volume fraction, height, width, depth, trabecular thickness, and anisotropy were quantified from midplanar micro-CT sections in 3 dimensions. Midsagittal PSB histologic sections stained with H&E and Safranin O/Fast Green were used to determine the ratio of ossification center to cartilage template size and to describe the formation and development of the cartilage template, ossification center, spherical growth plate, articular cartilage, and entheses. Mineralization of equine PSBs is associated with cartilage canals and a spherical growth plate that undergoes endochondral ossification during the late gestation to early postgestational period. The apical, flexor, basilar, and articular ossification fronts demonstrate morphologic variability. Structural organization of the articular cartilage and entheses occurs concurrently with the development of an underlying plate of compact bone. At 540 days postgestation, the fibrocartilaginous entheses of the flexor cortex of the PSB had yet to mature. Equine PSBs mineralize predominantly by endochondral ossification during the late gestation to early postgestational period. Mineralization precedes maturation of the articular cartilage and fibrocartilaginous entheses. The postgestational maturation of the PSB and its surrounding tissues may predispose young horses to developing lesions at these sites, such as apical avulsion fractures, warranting further investigation.

Three-dimensional gas-foamed scaffolds decorated with metal phenolic networks for cartilage regeneration

Inflammation is a major impediment to the healing of cartilage injuries, yet bioactive scaffolds suitable for cartilage repair in inflammatory environments are extremely rare. Herein, we utilized electrospinning to fabricate a two-dimensional nanofiber scaffold (2DS), which was then subjected to gas foaming to obtain a three-dimensional scaffold (3DS). 3DS was modified with metal phenolic networks (MPNs) composed of epigallocatechin gallate (EGCG) and strontium ions (Sr2+) to afford a MPNs-modified 3D scaffold (3DS-E). Gas-foamed scaffold exhibited multilayered structure conducive to cellular infiltration and proliferation. Compared to other groups, 3DS-E better preserved chondrocytes under interleukin (IL)-1β induced inflammatory environment, showing less apoptosis of chondrocytes and higher expression of cartilage matrix. Additionally, 3DS-E facilitated the regeneration of more mature cartilage in vivo, reduced cell apoptosis, and decreased the expression of pro-inflammatory cytokines.Taken together, 3DS-E may offer an ideal candidate for cartilage regeneration.

Involvement of mitogen- and stress-activated protein kinase 1 in BMP-6–induced chondrocyte differentiation

Bone morphogenetic proteins (BMPs) are involved in several cellular responsive actions, such as development, cell differentiation, and apoptosis, via their specific transmembrane receptors. In particular, BMPs promote the differentiation and maturation of bone and cartilage from mesenchymal stem cells. Based on comprehensive analyses performed with a large number of antibodies, mitogen- and stress-activated protein kinase (MSK)1 was found to be immediately phosphorylated in the mouse chondrocyte precursor cell line, ATDC5, upon BMP-6 stimulation. The overexpression and knockdown of MSK1 in ATDC5 cells also enhanced and suppressed BMP-6-induced chondrocyte differentiation, respectively. Similar to ATDC5 cells, an exvivo organ culture system using mouse embryonic metatarsal bones also demonstrated that BMP-6-mediated MSK1 activation might play a role in chondrocyte differentiation. Using several inhibitors, the p38 kinase pathway was confirmed to be implicated in BMP-6-induced phosphorylation of MSK1. Furthermore, MSK1 mutants lacking kinase activities and those lacking serine/threonine residues targeted by p38 kinase severely impaired their ability to potentiate BMP-6-induced chondrogenic differentiation of ATDC5 cells. Interestingly, a loss-of-function study for Smad4 perturbed BMP-6-induced phosphorylation of p38 kinase to inhibit BMP-6-mediated chondrocyte differentiation via MSK1 activation. Overall, both Smad-dependent and independent pathways require BMP-6-induced chondrocyte differentiation via MSK1 activation in ATDC5 cells.

Arthroscopic Shaver-based Harvest of Minced Cartilage Results in Reduced Chondrocyte Viability and Reduced Quality of Cartilaginous Repair Tissue Compared With Open Harvest and Conventional Fragmentation

To characterize and compare the quality of regenerative cartilage tissue (ReCT) after conventional minced cartilage (CMC) and arthroscopic minced cartilage (AMC), in terms of cell viability, gene expression, and matrix synthesis and to investigate the influence of different shaver types. Chondral tissue was harvested from the knees of 8 porcine donors. Porcine specimens were euthanized one day before harvest. AMC was created with 2 shaver blades in 2 operating modes (oscillating vs forward) and compared with a scalpel-fragmented CMC control. Before histologic analysis, 50% of the tissue was digested to prevent dedifferentiation of chondrocytes to fibroblasts. Cells were cultured and analyzed for cell viability, gene expression of cartilage-specific markers (aggrecan [ACAN], collagen type II, alpha1 [COL2A1], collagen type I, alpha1 [COL1A1], fibronectin-1 [FN1]), and matrix synthesis (Alcian-blue). AMC tissue contained fewer viable chondrocytes (41%-54% vs 91%; P= .001-.048) compared with CMC. After culture, CMC showed greater expressions of ACAN (27 virtual copy numbers [VCN]/housekeeping gene) and COL2A1 (30 VCN) compared with AMC (ACAN 2-9 VCN, COL2A1 2-7 VCN, P= .001-.039). AMC presented greater expressions of COL1A1 (9-21 VCN) and FN1 (12-17 VCN) than CMC (1 and 6 VCN, P= .001-.050). The signal intensity of the cartilage matrix formed by CMC (86/mm2) was greater than by AMC (7-10 mm2, P= .001-.032). CMC contained high numbers of viable chondrocytes, resulting in high-quality, hyaline-like ReCT. In contrast, AMC showed impaired chondrocyte quantity and viability, showing greater expressions of fibroblast markers and a decreased formation of mature cartilage matrix in porcine samples. The high chondrogenic potential of CMC to form hyaline-like ReCT was not confirmed for AMC. On the basis of our findings, arthroscopic harvest of minced cartilage leads to reduced chondrocyte viability and ReCT quality. Accordingly, CMC and AMC cannot be regarded as synonymous techniques, as arthroscopic techniques seem to be less efficacious.

Fibronectin isoforms promote postnatal skeletal development

Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months.We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-β1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation.In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.

Core-shell structured nanomembrane with sequential immune and vascular isolation effects followed by chondrogenic induction to promote stable stem cell-based subcutaneous cartilage regeneration in large animals

Bone marrow stem cell (BMSC)-regenerated cartilage (BRC) shows promising prospects for clinically repairing cartilage defects. However, when subcutaneously implanted into immunocompetent large animals, BRC is prone to ossification due to vascular invasion and inflammatory infiltration, compromising its chondrogenic stability. To address this challenge, we developed a core–shell structured KGN/Gel@Cur/PLCL nanomembrane using coaxial electrospinning technology to enhance the chondrogenic stability of BRC in subcutaneous niche. The shell layer comprises curcumin (Cur), known for its potent immunomodulatory and anti-angiogenic effects, and poly(L-lactide-co-caprolactone) (PLCL). The core layer comprises Kartogenin (KGN), a recognized chondrogenic inducer, and gelatin (Gel). This nanomembrane encapsulated BRCs induced from goat BMSCs in vitro and was subcutaneously implanted into autologous goats. The nanomembrane’s physical barrier and Cur release from the shell layer prevented vascular invasion and inflammatory infiltration. Additionally, KGN release from the core layer maintained the BRC’s chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and preserved its chondrogenic stability. chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and maintained its chondrogenic stability. Overall, this study highlights the effectiveness of the core–shell structured KGN/Gel@Cur/PLCL nanomembrane in providing sequential immune and vascular isolation, followed with chondrogenic induction, to prevent BRC ossification and preserve the chondrogenic stability of subcutaneously implanted BRC in large animal. This is crucial for the clinical translation of stem cell therapy into subcutaneous cartilage repair.

The proteasome subunit psmb1 is essential for craniofacial cartilage maturation and morphogenesis.

Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms, including lower jaw malformations; however, the underlying mechanisms are unknown. Here, we used a zebrafish proteasome subunit β 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibited a flattened ceratohyal and smaller Meckel's and palatoquadrate cartilages. Ceratohyal flattening was a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons was similarly perturbed. psmb1 mutants lacked the hyohyal muscles, and craniofacial tendons were shortened and disorganized. We additionally identified a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 was required in both tissue-autonomous and nonautonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.

A regenerative approach for temporomandibular joint repair: An invitro and exvivo study.

Current clinical approaches to regenerate temporomandibular joint (TMJ) articulating cartilage defects only treat the symptoms (i.e. pain and dysfunction) and do not seek to restore joint integrity for long-term relief. Therefore, we investigated a novel self-assembling tissue-engineered cartilage to overcome this significant clinical issue for TMJ regenerative purposes. Examine the maturation of dynamic self-regenerating cartilage (dSRC) using auricular chondrocytes and evaluate a novel combinatorial approach with fractional laser treatment and dSRC implantation for TMJ cartilage repair. A suspension of 107 freshly harvested rabbit ear chondrocytes was cultured under a continuous reciprocating motion to form the dSRC. After 2, 4 and 8 weeks of culture, dSRC samples were stained with H&E, Safranin-O and Toluidine Blue. Immunohistochemistry (IHC) was performed for collagens type I and II. Channels (300-500 μm diameter and 1.2-1.5 mm depth) were created in six freshly harvested condyles using a fractional Erbium laser. Two groups were tested: dSRC in a laser-ablated lesion (experimental) and an empty laser-ablated channel (control). TMJ condyles were cultured for up to 8 weeks and analysed as described above. H&E staining showed a high cell density in dSRC compared to native cartilage. All dSRC groups demonstrated intense Safranin-O staining, indicating high glycosaminoglycan (GAG) production and intense Toluidine Blue staining showed high proteoglycan content. IHC confirmed that dSRC consisted predominantly of collagen type II. The experimental group showed improved cartilage repair at both time points compared to the empty channels. dSRC viability and successful matrix formation were demonstrated invitro. The combination of fractional laser ablation and dSRC implantation enhanced cartilage repair.

Influence of DNA-methylation at multiple stages of limb chondrogenesis

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

Expression of Chondrogenic Potential Markers in Cultured Chondrocytes from the Human Knee Joint.

While substantial progress has been made in engineering cartilaginous constructs for animal models, further research is needed to translate these methodologies for human applications. Evidence suggests that cultured autologous chondrocytes undergo changes in phenotype and gene expression, thereby affecting their proliferation and differentiation capacity. This study was designed to evaluate the expression of chondrogenic markers in cultured human articular chondrocytes from passages 3 (P3) and 7 (P7), beyond the current clinical recommendation of P3. Cultured autologous chondrocytes were passaged from P3 up to P7, and quantitative polymerase chain reaction (qPCR) was used to assess mRNA expression of chondrogenic markers, including collagen type I (COLI), collagen type II (COLII), aggrecan (AGG), bone morphogenetic protein 4 (BMP4), transcription factor SOX-9 (SOX9), proteoglycan 4 (PGR4), and transformation-related protein 53 (p53), between P3 and P7. Except for AGG, no significant differences were found in the expression of markers between passages, suggesting the maintenance of chondrogenic potential in cultured chondrocytes. Differential expression identified between SOX9 and PGR4, as well as between COLI and SOX9, indicates that differences in chondrogenic markers are present between age groups and sexes, respectively. Overall, expression profiles of younger and male chondrocytes exhibit conversion of mature cartilage characteristics compared to their counterparts, with signs of dedifferentiation and loss of phenotype within-group passaging. These results may have implications in guiding the use of higher passaged chondrocytes for engineering constructs and provide a foundation for clinical recommendations surrounding the repair and treatment of articular cartilage pathology in both sexes.

Advances in Polysaccharides for Cartilage Tissue Engineering Repair: A Review.

Cartilage repair has been a significant challenge in orthopedics that has not yet been fully resolved. Due to the absence of blood vessels and the almost cell-free nature of mature cartilage tissue, the limited ability to repair cartilage has resulted in significant socioeconomic pressures. Polysaccharide materials have recently been widely used for cartilage tissue repair due to their excellent cell loading, biocompatibility, and chemical modifiability. They also provide a suitable microenvironment for cartilage repair and regeneration. In this Review, we summarize the techniques used clinically for cartilage repair, focusing on polysaccharides, polysaccharides for cartilage repair, and the differences between these and other materials. In addition, we summarize the techniques of tissue engineering strategies for cartilage repair and provide an outlook on developing next-generation cartilage repair and regeneration materials from polysaccharides. This Review will provide theoretical guidance for developing polysaccharide-based cartilage repair and regeneration materials with clinical applications for cartilage tissue repair and regeneration.

Hypoxic Conditions Modulate Chondrogenesis through the Circadian Clock: The Role of Hypoxia-Inducible Factor-1α.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor composed of an alpha and a beta subunit. HIF-1α is a master regulator of cellular response to hypoxia by activating the transcription of genes that facilitate metabolic adaptation to hypoxia. Since chondrocytes in mature articular cartilage reside in a hypoxic environment, HIF-1α plays an important role in chondrogenesis and in the physiological lifecycle of articular cartilage. Accumulating evidence suggests interactions between the HIF pathways and the circadian clock. The circadian clock is an emerging regulator in both developing and mature chondrocytes. However, how circadian rhythm is established during the early steps of cartilage formation and through what signaling pathways it promotes the healthy chondrocyte phenotype is still not entirely known. This narrative review aims to deliver a concise analysis of the existing understanding of the dynamic interplay between HIF-1α and the molecular clock in chondrocytes, in states of both health and disease, while also incorporating creative interpretations. We explore diverse hypotheses regarding the intricate interactions among these pathways and propose relevant therapeutic strategies for cartilage disorders such as osteoarthritis.

Osteochondral Allograft Transplantation in the Knee

Osteochondral injuries of the knee can be a frequent source of debilitating pain and dysfunction. Significant chondral (>1.5-2 cm2) lesions of the femoral condyles can be especially difficult to manage with nonsurgical measures. Fresh osteochondral allograft (OCA) transplantation has been shown to be a reliable surgical procedure to manage a wide array of high-grade focal chondral lesions, with or without subchondral bone involvement. OCA transplantation affords the transfer of a size-matched allograft of mature hyaline cartilage with its associated subchondral bony scaffold. Indications include primary or secondary management of large, high-grade chondral or osteochondral defects secondary to trauma, developmental malformation, osteonecrosis, or other focal degenerative disease. Contraindications include end-stage osteoarthritis, uncorrected malalignment, ligament or meniscus deficiency, and inflammatory joint disease. Improvements in surgical technique, allograft storage, and tissue availability have created more reproducible clinical results and increased chondrocyte viability. Long-term (>10 year) graft survival rates have been shown to be between 70% and 91%, and the procedure has been shown to be cost-effective based on cost per quality-adjusted life year. Finally, OCA transplantation has been shown to provide excellent return to play rate for athletes with medium-to-large cartilage lesions. OCA transplantation is therefore an important option in the treatment algorithm of articular cartilage injuries.

A Rare Case of Bronchogenic Cyst in a Newborn Baby

Bronchogenic cyst is a rare congenital anomaly of the formation of the pulmonary germ, a thin–walled formation filled with thick contents, which distinguishes it from aerial bronchial cysts. Clinically, a bronchogenic cyst can manifest itself as obstructive respiratory failure, swallowing disorders, and lead to recurrent purulent-­inflammatory processes. In this article, we present a clinical case of bronchogenic cyst in a newborn child, detected during pregnancy at the 2nd ultrasound screening and successfully operated on as planned. A 5-day-old boy was transferred to a surgical hospital for surgical treatment of cystic formation of the left lung S10 with dimensions 16.0×12.0×15.0 mm; the cyst contents were homogeneous, corresponding to the fluid. Surgical treatment was performed — the formation was separated from the parietal pleura and sent to the pathology department for histological examination. No complications were observed in the postoperative period. The macroscopically studied object was a three-­dimensional formation of gray-red color with a bluish tinge of irregular shape, dull, with a dark red fine sprinkling. A thin-walled cavity filled with cloudy jelly-like contents was determined on the incision. Microscopic description — a cyst lined with ciliated epithelium, partially flattened to cubic due to dystrophic changes, subepithelial islets of mature cartilage tissue and small groups of mucous glands. Thus, a bronchogenic cyst was morphologically verified. Differential diagnosis of bronchogenic cysts from inflammatory lung diseases (tuberculosis, abscess) with untimely diagnosis in the absence of clinical manifestations is practically impossible without histological examination. Therefore, prenatal diagnosis and morphological examination of the surgical material are crucial.

Enzymatic digestion does not compromise sliding-mediated cartilage lubrication

Articular cartilage's remarkable low-friction properties are essential to joint function. In osteoarthritis (OA), cartilage degeneration (e.g., proteoglycan loss and collagen damage) decreases tissue modulus and increases permeability. Although these changes impair lubrication in fully depressurized and slowly slid cartilage, new evidence suggests such relationships may not hold under biofidelic sliding conditions more representative of those encountered in vivo. Our recent studies using the convergent stationary contact area (cSCA) configuration demonstrate that articulation (i.e., sliding) generates interfacial hydrodynamic pressures capable of replenishing cartilage interstitial fluid/pressure lost to compressive loading through a mechanism termed tribological rehydration. This fluid recovery sustains in vivo-like kinetic friction coefficients (µk<0.02 in PBS and <0.005 in synovial fluid) with little sensitivity to mechanical properties in healthy tissue. However, the tribomechanical function of compromised cartilage under biofidelic sliding conditions remains unknown. Here, we investigated the effects of OA-like changes in cartilage mechanical properties, modeled via enzymatic digestion of mature bovine cartilage, on its tribomechanical function during cSCA sliding. We found no differences in sliding-driven tribological rehydration behaviors or µk between naïve and digested cSCA cartilage (in PBS or synovial fluid). This suggests that OA-like cartilage retains sufficient functional properties to support naïve-like fluid recovery and lubrication under biofidelic sliding conditions. However, OA-like cartilage accumulated greater total tissue strains due to elevated strain accrual during initial load application. Together, these results suggest that elevated total tissue strains—as opposed to activity-mediated strains or friction-driven wear—might be the key biomechanical mediator of OA pathology in cartilage. Statement of SignificanceOsteoarthritis (OA) decreases cartilage's modulus and increases its permeability. While these changes compromise frictional performance in benchtop testing under low fluid load support (FLS) conditions, whether such observations hold under sliding conditions that better represent the joints’ dynamic FLS conditions in vivo is unclear. Here, we leveraged biofidelic benchtop sliding experiments—that is, those mimicking joints’ native sliding environment—to examine how OA-like changes in mechanical properties effect cartilage's natural lubrication. We found no differences in sliding-mediated fluid recovery or kinetic friction behaviors between naïve and OA-like cartilage. However, OA-like cartilage experienced greater strain accumulation during load application, suggesting that elevated tissue strains (not friction-driven wear) may be the primary biomechanical mediator of OA pathology.

Proteoglycan inhibition of canonical BMP-dependent cartilage maturation delays endochondral ossification.

During endochondral ossification, chondrocytes secrete a proteoglycan (PG)-rich extracellular matrix that can inhibit the process of cartilage maturation, including expression of Ihh and Col10a1. Because bone morphogenetic proteins (BMPs) can promote cartilage maturation, we hypothesized that cartilage PGs normally inhibit BMP signalling. Accordingly, BMP signalling was evaluated in chondrocytes of wild-type and PG mutant (fam20b-/-) zebrafish and inhibited with temporal control using the drug DMH1 or an inducible dominant-negative BMP receptor transgene (dnBMPR). Compared with wild type, phospho-Smad1/5/9, but not phospho-p38, was increased in fam20b-/- chondrocytes, but only after they secreted PGs. Phospho-Smad1/5/9 was decreased in DMH1-treated or dnBMPR-activated wild-type chondrocytes, and DMH1 also decreased phospho-p38 levels. ihha and col10a1a were decreased in DMH1-treated or dnBMPR-activated chondrocytes, and less perichondral bone formed. Finally, early ihha and col10a1a expression and early perichondral bone formation of fam20b mutants were rescued with DMH1 treatment or dnBMPR activation. Therefore, PG inhibition of canonical BMP-dependent cartilage maturation delays endochondral ossification, and these results offer hope for the development of growth factor therapies for skeletal defects of PG diseases.

MULTILAYER DUAL-POROSITY 3D-PRINTED SCAFFOLDS TO RECREATE THE ANISOTROPIC MICROENVIRONMENT OF THE CARTILAGE

Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure.Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed.Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour.In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage.Acknowledgments: This work was supported by the EMAKIKER grant.

Changes in tissue sodium concentration and sodium relaxation times during the maturation of human knee cartilage: Ex vivo 23 Na MRI study at 10.5 T.

To evaluate the influence of skeletal maturation on sodium (23 Na) MRI relaxation parameters and the accuracy of tissue sodium concentration (TSC) quantification in human knee cartilage. Twelve pediatric knee specimens were imaged with whole-body 10.5 T MRI using a density-adapted 3D radial projection sequence to evaluate 23 Na parameters: B1 + , T1 , biexponential , and TSC. Water, collagen, and sulfated glycosaminoglycan (sGAG) content were calculated from osteochondral biopsies. The TSC was corrected for B1 + , relaxation, and water content. The literature-based TSC (TSCLB ) used previously published values for corrections, whereas the specimen-specific TSC (TSCSP ) used measurements from individual specimens. 23 Na parameters were evaluated in eight cartilage compartments segmented on proton images. Associations between 23 Na parameters, TSCLB - TSCSP difference, biochemical content, and age were determined. From birth to 12 years, cartilage water content decreased by 18%; collagen increased by 59%; and sGAG decreased by 36% (all R2 ≥ 0.557). The short ( ) decreased by 72%, and the signal fraction relaxing with ( ) increased by 55% during the first 5 years but remained relatively stable after that. TSCSP was significantly correlated with sGAG content from biopsies (R2 = 0.739). Depending on age, TSCLB showed higher or lower values than TSCSP . The TSCLB - TSCSP difference was significantly correlated with (R2 = 0.850), (R2 = 0.651), and water content (R2 = 0.738). TSC and relaxation parameters measured with 23 Na MRI provide noninvasive information about changes in sGAG content and collagen matrix during cartilage maturation. Cartilage TSC quantification assuming fixed relaxation may be feasible in children older than 5 years.

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.

Double-edged role of mechanical stimuli and underlying mechanisms in cartilage tissue engineering.

Mechanical stimuli regulate the chondrogenic differentiation of mesenchymal stem cells and the homeostasis of chondrocytes, thus affecting implant success in cartilage tissue engineering. The mechanical microenvironment plays fundamental roles in the maturation and maintenance of natural articular cartilage, and the progression of osteoarthritis Hence, cartilage tissue engineering attempts to mimic this environment in vivo to obtain implants that enable a superior regeneration process. However, the specific type of mechanical loading, its optimal regime, and the underlying molecular mechanisms are still under investigation. First, this review delineates the composition and structure of articular cartilage, indicating that the morphology of chondrocytes and components of the extracellular matrix differ from each other to resist forces in three top-to-bottom overlapping zones. Moreover, results from research experiments and clinical trials focusing on the effect of compression, fluid shear stress, hydrostatic pressure, and osmotic pressure are presented and critically evaluated. As a key direction, the latest advances in mechanisms involved in the transduction of external mechanical signals into biological signals are discussed. These mechanical signals are sensed by receptors in the cell membrane, such as primary cilia, integrins, and ion channels, which next activate downstream pathways. Finally, biomaterials with various modifications to mimic the mechanical properties of natural cartilage and the self-designed bioreactors for experiment in vitro are outlined. An improved understanding of biomechanically driven cartilage tissue engineering and the underlying mechanisms is expected to lead to efficient articular cartilage repair for cartilage degeneration and disease.