Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets.

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets' chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets' initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets' characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

Stem Cells and Extrusion 3D Printing for Hyaline Cartilage Engineering.

Hyaline cartilage is deficient in self-healing properties. The early treatment of focal cartilage lesions is a public health challenge to prevent long-term degradation and the occurrence of osteoarthritis. Cartilage tissue engineering represents a promising alternative to the current insufficient surgical solutions. 3D printing is a thriving technology and offers new possibilities for personalized regenerative medicine. Extrusion-based processes permit the deposition of cell-seeded bioinks, in a layer-by-layer manner, allowing mimicry of the native zonal organization of hyaline cartilage. Mesenchymal stem cells (MSCs) are a promising cell source for cartilage tissue engineering. Originally isolated from bone marrow, they can now be derived from many different cell sources (e.g., synovium, dental pulp, Wharton's jelly). Their proliferation and differentiation potential are well characterized, and they possess good chondrogenic potential, making them appropriate candidates for cartilage reconstruction. This review summarizes the different sources, origins, and densities of MSCs used in extrusion-based bioprinting (EBB) processes, as alternatives to chondrocytes. The different bioink constituents and their advantages for producing substitutes mimicking healthy hyaline cartilage is also discussed.

Cultivo condral in vitro bajo estímulos de compresión y cizallamiento. De las células troncales mesenquimales al cartílago hialino / In vitro chondral culture under compression and shear stimuli. From mesenchymal stem cells to hyaline cartilage

INTRODUCCIÓN: La creación in vitro de cartílago hialino articular supone un reto, ya que, a día de hoy, no se ha conseguido la síntesis ex vivo de un tejido estructurado con las mismas propiedades biomecánicas e histológicas del cartílago articular. Para simular las condiciones fisiológicas hemos diseñado un sistema de cultivo in vitro que reproduce el movimiento articular. MATERIAL Y MÉTODO: Hemos desarrollado un biorreactor de cultivo celular que imprime un estímulo mecánico sobre una matriz de elastina en la que están embebidas células troncales mesenquimales (MSC). La primera fase de estudio corresponde al desarrollo de un biorreactor para cultivo de cartílago hialino y la comprobación de la viabilidad celular en la matriz de elastina en ausencia de estímulo. La segunda fase del estudio engloba el cultivo de MSC bajo estímulo mecánico y el análisis del tejido resultante. RESULTADOS: Tras el cultivo bajo estímulo mecánico no obtuvimos tejido hialino por falta de celularidad y desestructuración de la matriz. CONCLUSIÓN: El patrón de estímulo utilizado no ha resultado efectivo para la generación de cartílago hialino, por lo que se deberán explorar otras combinaciones en futuras investigaciones

INTRODUCTION: The in vitro creation of hyaline joint cartilage is a challenge since, to date, the ex vivo synthesis of a structured tissue with the same biomechanical and histological properties of the joint cartilage has not been achieved. To simulate the physiological conditions we have designed an in vitro culture system that reproduces joint movement. MATERIAL AND METHOD: We have developed a cell culture bioreactor that prints a mechanical stimulus on an elastin matrix, in which mesenchymal stem cells (MSC) are embedded. The first phase of study corresponds to the development of a bioreactor for hyaline cartilage culture and the verification of cell viability in the elastin matrix in the absence of stimulus. The second phase of the study includes the MSC culture under mechanical stimulus and the analysis of the resulting tissue. RESULTS: After culture under mechanical stimulation we did not obtain hyaline tissue due to lack of cellularity and matrix destructuring. CONCLUSION: The stimulus pattern used has not been effective in generating hyaline cartilage, so other combinations should be explored in future research

The Janus Role of Adhesion in Chondrogenesis.

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell-cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell-substrate adhesion in the tissue engineering strategies for cartilage repair.

The chondrogenic differentiation potential of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are particularly promising for tissue engineering (TE) due to the ease of their isolation procedure, great expansion potential and capability to differentiate towards several cell types of the mesodermal, ectodermal and endodermal lineages. Although several studies hint that DPSCs exhibit potential for cartilage tissue formation, the chondrogenic potential of DPSCs has only been marginally explored. Thus, the aim of the present study was to closely investigate the chondrogenic differentiation capacity of DPSCs for TE applications. More specifically, the potential of DPSCs for engineering hyaline and fibrous cartilage was determined. DPSCs obtained from 7 human molars were expanded and chondrogenically differentiated in a 3D pellet culture model. After 21 d of differentiation with chondrogenic stimuli, DPSCs displayed glycosaminoglycan, aggrecan and limited collagen type II deposition. Cells presented an elongated morphology and produced a collagen-rich extracellular matrix, with a predominance of collagen type I in most of the samples, a characteristic of fibrous cartilage tissue. Variations in the administration periods of several chondro-inductive growth factors, including transforming growth factor beta 3, bone morphogenetic protein-2, -6, -7 and insulin-like growth factor-1, did not increase glycosaminoglycan or collagen type II deposition, typical markers of hyaline cartilage tissue. Furthermore, DPSCs could not be stimulated to go into hypertrophic chondrogenesis. These results indicated that under a large variety of chondro-inductive culture conditions, DPSCs could form fibrocartilaginous tissues but not hyaline cartilage. Thus, DPSCs represent a valuable cell source for the regeneration of fibrocartilage in joints.

Effects of Micronized Cartilage Matrix on Cartilage Repair in Osteochondral Lesions of the Talus.

BACKGROUND: The repair of osteochondral lesions remains a challenge due to its poor vascularity and limited healing potential. Micronized cartilage matrix (MCM) is dehydrated, decellularized, micronized allogeneic cartilage matrix that contains the components of native articular tissue and is hypothesized to serve as a scaffold for the formation of hyaline-like tissue. Our objective was to demonstrate in vitro that the use of MCM combined with mesenchymal stem cells (MSCs) can lead to the formation of hyaline-like cartilage tissue in a single-stage treatment model. DESIGN: In group 1 (no wash), 250 µL MCM was reconstituted in 150 µL Dulbecco's phosphate-buffered saline (DPBS) for 5 minutes. Group 2 (saline wash) included 250 µL MCM washed in 20 mL DPBS for 30 minutes, then aspirated to remove all DPBS and reconstituted in 150 µL DPBS. Group 3 (serum wash): 250µL MCM washed in 20 mL DPBS for 30 minutes, then aspirated and reconstituted in 150 µL fetal bovine serum. Each group was then added to 50 µL solution of MSC suspended in DPBS at a concentration of 1.2 × 106 cells/350 µL. After 3 weeks, the defects were extracted and sectioned to perform viability and histologic analyses. RESULTS: Stem cells without rehydration of the MCM showed almost no viability whereas near complete cell viability was seen after rehydration with serum or saline solution, ultimately leading to chondrogenic differentiation and adhesion to the MCM particles. CONCLUSION: We have shown in this proof-of-concept in vitro study that MCM can serve as a scaffold for the growth of cartilage tissue for the treatment of osteochondral lesions.

T140 Inhibits Apoptosis and Promotes Proliferation and Matrix Formation Through the SDF-1/CXC Receptor-4 Signaling Pathway in Endplate Chondrocytes of the Rat Intervertebral Discs.

BACKGROUND: Cartilaginous endplate (CEP), a thin layer of hyaline cartilage located between the vertebral endplate and nucleus pulposus, transports the nutrient into the disc. The objective of this study was to evaluate the influence of T140 (polyphemusin II-derived peptide) on the CEP cell growth, apoptosis, and the matrix formation via the stromal cell-derived factor-1 (SDF-1)/cysteine X cysteine (CXC) receptor-4 (CXCR4) signaling pathway. METHODS: Sprague-Dawley rats were euthanized by cervical dislocation and dissected for the isolation and the appraisal of CEP cells that were extracted from the endplate in rat intervertebral discs and were then added with different concentrations of reagents (SDF-1 and T140). The effect of T140 on CEP cell proliferation and apoptosis were analyzed. The messenger RNA (mRNA) and protein expressions of CXCR4, prominin-1, proteoglycans, type II collagen, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein were analyzed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. RESULTS: T140 promoted the proliferation of CEP cells and inhibited the apoptosis of CEP cells. Additionally, T140 suppressed the mRNA and protein expression of CXCR4, prominin-1, and Bcl-2 associated X protein, and increased the mRNA and protein expression of proteoglycans, type II collagen, and Bcl-2. CONCLUSIONS: T140 promotes the proliferation and matrix formation and inhibits the apoptosis of CEP cells by blocking the SDF-1/CXCR4 signaling pathway in vitro, which provides a certain therapeutic effect on the degeneration of intervertebral discs.

In Vivo Formation of Stable Hyaline Cartilage by Naïve Human Bone Marrow Stromal Cells with Modified Fibrin Microbeads.

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

Hyaline cartilage next generation implants from adipose-tissue-derived mesenchymal stem cells: Comparative study on 3D-printed polycaprolactone scaffold patterns.

We used additive manufacturing to fabricate 3D-printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose-tissue-derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three-dimensional honeycomb-like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

OBJECTIVE: Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. DESIGN: Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. RESULTS: It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. CONCLUSION: The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

A 3-dimensional bioprinted tracheal segment implant pilot study: Rabbit tracheal resection with graft implantation.

OBJECTIVES: Surgical reconstruction of tracheal disease has expanded to include bioengineering and three dimensional (3D) printing. This pilot study investigates the viability of introducing a living functional tracheal replacement graft in a rabbit animal model. METHODS: Seven New Zealand White rabbits were enrolled and six completed participation (one intraoperative mortality). Tracheal replacement grafts were created by impregnating 3D printed biodegradable polycaprolactone (PCL) tracheal scaffolds with rabbit tracheal hyaline chondrocytes. 2 cm of native trachea was resected and the tracheal replacement graft implanted. Subjects were divided into two equal groups (n = 3) that differed in their time of harvest following implantation (three or six weeks). Tracheal specimens were analyzed with intraluminal telescopic visualization and histopathology. RESULTS: The two groups did not significantly differ in histopathology or intraluminal diameter. All sections wherein the implant telescoped over native trachea (anastomotic ends) contained adequate hyaline cartilage formation (i.e. chondrocytes within lacuna, surrounding extracellular matrix, and strong Safranin O staining). Furthermore, the PCL scaffold was surrounded by a thin layer of fibrous tissue. All areas without membranous coverage contained inadequate or immature cartilage formation with inflammation. The average intraluminal stenosis was 83.4% (range 34.2-95%). CONCLUSIONS: We report normal cartilage growth in a tracheal replacement graft when chondrocytes are separated from the tracheal lumen by an intervening membrane. When no such membrane exists there is a propensity for inflammation and stenosis. These findings are important for future construction and implantation of tracheal replacement grafts. LEVEL OF EVIDENCE: Not applicable: this is an in vivo animal trial.

Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential via modulation of culture conditions and Wnt/ß-catenin pathway.

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate articular cartilage, but current chondroinduction protocols, commonly using transforming growth factor-ß (TGFß), lead to concomitant chondrocytic hypertrophy with ossification risk. Here, we showed that a 14-day culture of MSC-laden hyaluronic acid hydrogel in the presence of TGFß, followed by 7 days culture in TGFß-free medium, with the supplement of Wnt/ß-catenin inhibitor XAV939 from day 10-21, resulted in significantly reduced hypertrophy phenotype. The stability of the hyaline phenotype of the MSC-derived cartilage, generated with a standard protocol (Control) or the optimized (Optimized) method developed in this study, was further examined through intramuscular implantation in nude mice. After 4 weeks, constructs from the Control group showed obvious mineralization; in contrast, the Optimized group displayed no signs of mineralization, and maintained cartilaginous histology. Further analysis showed that TGFß treatment time affected p38 expression, while exposure to XAV939 significantly inhibited P-Smad 1/5 level, which together resulted in decreased level of Runx2. These findings suggest a novel treatment regimen to generate hyaline cartilage from human MSCs-loaded scaffolds, which have a minimal risk of eliciting endochondral ossification.

Injectable glycosaminoglycan-protein nano-complex in semi-interpenetrating networks: A biphasic hydrogel for hyaline cartilage regeneration.

Articular hyaline cartilage regeneration remains challenging due to its less intrinsic reparability. The study develops injectable biphasic semi-interpenetrating polymer networks (SIPN) hydrogel impregnated with chondroitin sulfate (ChS) nanoparticles for functional cartilage restoration. ChS loaded zein nanoparticles (∼150nm) prepared by polyelectrolyte-protein complexation were interspersed into injectable SIPNs developed by blending alginate with poly(vinyl alcohol) and calcium crosslinking. The hydrogel exhibited interconnected porous microstructure (39.9±5.8µm pore diameter, 57.7±5.9% porosity), 92% swellability and >350Pa elastic modulus. Primary chondrocytes compatibility, chondrocyte-matrix interaction with cell-cell clustering and spheroidal morphology was demonstrated in ChS loaded hydrogel and long-term (42days) proliferation was also determined. Higher fold expression of cartilage-specific genes sox9, aggrecan and collagen-II was observed in ChS loaded hydrogel while exhibiting poor expression of collagen-I. Immunoblotting of aggregan and collagen II demonstrate favorable positive influence of ChS on chondrocytes. Thus, the injectable biphasic SIPNs could be promising composition-mimetic substitute for cartilage restoration at irregular defects.

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1.

Articular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-ß1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-ß1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model.

Sol-gel derived lithium-releasing glass for cartilage regeneration.

Wnt-signalling cascade is one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO2-Li2O sol-gel derived glass for controlled delivery of lithium, a known glycogen synthase kinase-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation in an in vitro 3D pellet culture model is reported. Dissolution products that contained 5 mM lithium and 3.5 mM silicon were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-ß3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

Fresh osteochondral allografts-procurement and tissue donation in Europe.

Fresh osteochondral allografts are a well-established treatment for large, full-thickness cartilage defects. The clinical outcome for carefully selected patients is very favorable, especially for the young and active and graft survival up to 25 years has been described in the literature. Furthermore, a high patient satisfaction rate has been reported, but the biggest obstacle to overcome is the availability of tissue for transplantation. Large fresh bone allografts for cartilage damage repair only can be harvested from organ donors following organ removal or cadaveric donors, preferably in the setting of an operation room to minimize possible contamination of the tissue. Apart from the logistic challenges this entails, an experienced recovery team is needed. Furthermore, the public as well as medical staff is much less aware of the possibility and requirements of tissue donation than organ donation and families of deceased are rarely approached for bone and cartilage donation. This review aims to highlight the current situation of organ and tissue donation in Europe with special focus on the processing of bones and possible safety and quality concerns. We analyze what may prevent consent and what might be done to improve the situation of tissue donation.

Vascular Canals in Permanent Hyaline Cartilage: Development, Corrosion of Nonmineralized Cartilage Matrix, and Removal of Matrix Degradation Products.

Core areas in voluminous pieces of permanent cartilage are metabolically supplied via vascular canals (VCs). We studied cartilage corrosion and removal of matrix degradation products during the development of VCs in nose and rib cartilage of piglets. Conventional staining methods were used for glycosaminoglycans, immunohistochemistry was performed to demonstrate collagens types I and II, laminin, Ki-67, von Willebrand factor, VEGF, macrophage marker MAC387, S-100 protein, MMPs -2,-9,-13,-14, and their inhibitors TIMP1 and TIMP2. VCs derived from connective tissue buds that bulged into cartilage matrix ("perichondrial papillae", PPs). Matrix was corroded at the tips of PPs or resulting VCs. Connective tissue stromata in PPs and VCs comprised an axial afferent blood vessel, peripherally located wide capillaries, fibroblasts, newly synthesized matrix, and residues of corroded cartilage matrix (collagen type II, acidic proteoglycans). Multinucleated chondroclasts were absent, and monocytes/macrophages were not seen outside the blood vessels. Vanishing acidity characterized areas of extracellular matrix degradation ("preresorptive layers"), from where the dismantled matrix components diffused out. Leached-out material stained in an identical manner to intact cartilage matrix. It was detected in the stroma and inside capillaries and associated downstream veins. We conclude that the delicate VCs are excavated by endothelial sprouts and fibroblasts, whilst chondroclasts are specialized to remove high volumes of mineralized cartilage. VCs leading into permanent cartilage can be formed by corrosion or inclusion, but most VCs comprise segments that have developed in either of these ways. Anat Rec, 300:1067-1082, 2017. © 2016 Wiley Periodicals, Inc.

Possible role of the Ec peptide of IGF­1Ec in cartilage repair.

The Ec peptide (PEc) of insulin-like growth factor 1 Ec (IGF-1Ec) induces human mesenchymal stem cell (hMSC) mobilization and activates extracellular signal­regulated kinase 1/2 (ERK 1/2) in various cells. The aim of the present study was to examine the effects of PEc on the mobilization and differentiation of hMSCs, as well as the possibility of its implementation in combination with transforming growth factor ß1 (TGF­ß1) for cartilage repair. The effects of the exogenous administration of PEc and TGF­ß1, alone and in combination, on hMSCs were assessed using a trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays and migration/invasion assays. It was determined that PEc is involved in the differentiation process of hMSCs towards hyaline cartilage. Treatment of hMSCs with either PEc, TGF­ß1 or both, demonstrated comparable cartilage matrix deposition. Furthermore, treatment with PEc in combination with TGF­ß1 was associated with a significant increase in hMSC mobilization when compared with treatment with TGF­ß1 or PEc alone (P<0.05). Thus, PEc appears to facilitate in vitro hMSC mobilization and differentiation towards chondrocytes, enhancing the role of TGF­ß1.