Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling.

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

Performance of Colombian Silk Fibroin Hydrogels for Hyaline Cartilage Tissue Engineering.

The development and evaluation of scaffolds play a crucial role in the engineering of hyaline cartilage tissue. This work aims to evaluate the performance of silk fibroin hydrogels fabricated from the cocoons of the Colombian hybrid in the in vitro regeneration of hyaline cartilage. The scaffolds were physicochemically characterized, and their performance was evaluated in a cellular model. The results showed that the scaffolds were rich in random coils and ß-sheets in their structure and susceptible to various serine proteases with different degradation profiles. Furthermore, they showed a significant increase in ACAN, COL10A1, and COL2A1 expression compared to pellet culture alone and allowed GAG deposition. The soluble portion of the scaffold did not affect chondrogenesis. Furthermore, they promoted the increase in COL1A2, showing a slight tendency to differentiate towards fibrous cartilage. The results also showed that Colombian silk could be used as a source of biomedical devices, paving the way for sericulture to become a more diverse economic activity in emerging countries.

lncRNAs in mesenchymal differentiation.

In recent years, technological advances have revealed a large potential number of long noncoding RNAs (lncRNAs). Findings recognize lncRNAs as orchestrating molecules in a wide range of processes, at the transcriptional and posttranscriptional levels, although fewer studies address function. For differentiation, which consists of rearrangements in the gene expression profile and activation of stage- and cell type-dependent signaling mechanisms, the relevance of lncRNAs becomes crucial. The relationship between lncRNAs and key molecular factors in differentiation is strengthening; therefore the present review aims to comprehensively explain the role of lncRNAs in the signaling network involved in the main types of mesenchymal differentiation: adipogenesis, chondrogenesis, myogenesis, and osteogenesis. Notably, a step toward the integration of lncRNAs in the field of cell differentiation promises an exceptional impact.

Triiodothyronine does not influence in vitro chondrogenic differentiation of adipose tissue-derived stem cells from young female rat / Triiodotironina não influencia na diferenciação condrogênica de células tronco do tecido adipose de ratas jovens

The objective of this study was to investigate the in vitro action of triiodothyronine (T3) on the chondrogenic differentiation of adipose tissue-derived stem cells (ASCs) of female rats, with different time periods and doses. ASCs were extracted from female Wistar rats and were cultured in chondrogenic medium with and without the presence of T3. Five groups were established: 1) ASCs without T3; and 2,3,4,5) ASCs with 0.01, 1, 100 and 1,000 nM T3, respectively). After 7, 14 and 21 days, cell morphology, chondrogenic matrix formation, and expression of Sox9, aggrecan, collagen II, and collagen X were evaluated. The Student-Newman-Keuls test was used. ASCs showed CD54, CD73, and CD90 before chondrogenic differentiation. The hormone treatment did not alter chondrogenic matrix formation, Sox9 expression at 14 or 21 days, or expression of collagen II or collagen X at any time. However, the 0.01, 1, and 1000 nM T3 doses decreased Sox9 expression at 7 days. In conclusion, chondrogenic differentiation of ASCs of female rats is not influenced by T3.

O objetivo do presente trabalho foi verificar o efeito in vitro da triiodotironina (T3) na diferenciação condrogênica de células tronco mesenquimais do tecido adiposo (CTM-TA) de ratas, durante vários períodos e em várias doses. CTM-TA foram coletadas de ratas Wistar e cultivadas em meio condrogênico com ou sem a presença de T3. Constitui-se cinco grupos: 1) CTM-TA sem T3; e 2,3,4,5) CTM-TA com T3 (0,01; 1; 100 e 1000 nM, respectivamente). Após sete, 14 e 21 dias, foram avaliados morfologia celular, formação de matriz condrogênica e expressão de Sox9, agrecano, colágeno II e colágeno X. Para as análises foi utilizado o teste de Student Newman Keuls. CTM-TA expressaram CD54, CD73 e CD90 antes da diferenciação condrogênica. O tratamento hormonal não alterou a formação de matriz condrogênica e a expressão de Sox9 aos 14 e 21 dias e expressão dos colágenos II e X em nenhum dos períodos avaliados. No entanto, as doses de 0,01; 1 e 1000 nM T3 diminuíram a expressão de Sox9 aos 7 dias. Conclui-se que a diferenciação condrogênica de CTM-TA de ratas não é influenciada pela T3.

Potential effect of hyaluronic acid and triamcinolone acetate, alone or combined, on chondrogenic differentiation of mesenchymal stem cells / Efecto potencial del ácido hialurónico y del acetato de triamcinolona solos o combinados sobre la diferenciación condrogénica de células-tronco mesenquimales / Efeito potencial do ácido hialurônico e triancinolona acetonida, isolados e combinados, na diferenciação condrogênica de células-tronco mensenquimais

Abstract Background: Osteoarthritis is a complex degenerative disease with several factors contributing to joint damage. Objective: To compare the potential effect of hyaluronic acid (HA) and triamcinolone acetonide (TA), alone or combined, on the in vitro chondrogenic differentiation process of mesenchymal stem cells (MSCs). Methods: MSCs were divided into four groups: Control, HA, TA, and HA/TA combined. Each treatment group was cultured for 14 days in chondrogenic differentiation medium. The chondrogenic differentiation potential was assessed by histology and immunohistochemistry. Results: The HA and HA/TA-treated MSCs presented histological characteristics similar to native chondrocytes. The extracellular matrix (ECM) of TA-treated MSCs was compact and organized. Glycosaminoglycan staining was intense in Control, moderate in TA, slight in HA/TA, and undetectable in HA. Type II collagen immunoreactivity was high in the TA-treated ECM and MSCs. Conclusions: Histological analysis shows that HA influences morphological development similar to chondrocytes of the MSCs, but with low expression of specific cartilage molecules. The TA promotes formation of a compact and organized ECM.

Resumen Antecedentes: La osteoartritis es una enfermedad degenerativa compleja en la cual varios factores contribuyen al daño articular. Objetivo: Comparar el efecto del ácido hialurónico (HA) y acetónido de triamcinolona (TA), solos o en combinación, en el proceso de diferenciación condrogénica in vitro de células madre mesenquimales (MSCs). Métodos: Las MSCs fueron divididas en cuatro grupos: Control, HA, TA y HA/TA, y cultivadas por 14 días en medio de diferenciación condrogénica para cada tratamiento. El potencial de diferenciación condrogénica fue analizado por medio de histología e inmunohistoquímica. Resultados: Las MSCs tratadas con HA y HA/TA, presentaron características histológicas similares a los condrocitos nativos, y la matriz extracelular (ECM) de MSCs tratadas con TA fue más compacta y organizada. La tinción de glicosaminoglicanos fue intensa en el Control, moderada en TA, ligera en HA/TA, y sin tinción en HA. La inmunoreactividad para colágeno tipo II fue más alta en las MSCs y ECM tratadas con TA. Conclusión: El análisis histológico muestra que el HA influencia un desarrollo morfológico similar a los condrocitos de las MSCs, pero con baja expresión de moléculas específicas de cartílago. La TA promueve la formación de una ECM compacta y organizada.

Resumo Antecedentes: A osteoartrite é uma doença degenerativa complexa, na qual vários fatores contribuem ao dano articular. Objetivo: Comparar o efeito do ácido hialurônico (HA) e Triancinolona acetonida (TA), só ou combinado no processo de diferenciação condrogênica in vitro de células tronco mesenquimais (MSCs). Métodos: MSCs foram divididas em 4 grupos: Controle, HA, TA y HA/TA e cultivadas por 14 dias com meio de diferenciação condrogênica e seus respectivos tratamentos. O potencial de diferenciação condrogênica foi acessado por meio de histologia e imunohistoquímica. Resultados: Histologicamente, MSCs tratadas com HA e HA/TA apresentaram características semelhantes de condrócitos nativos, e a matriz extracelular de MSCs tratadas com TA foi mais compacta e organizada. A coloração para glicosaminoglicanos foi intensa no Controle, moderada no TA, leve no HA/TA e sem coloração com HA. Para os grupos tratamento, a imunoreatividade para colágeno tipo II foi maior nas células e matriz extracelular tratadas com TA. Conclusão: Mediante análise histológica, o HA influenciou o desenvolvimento morfológico semelhante a condrócitos das MSCs, mas com baixa expressão de moléculas específicas de cartilagem. A TA promoveu a formação de uma matriz extracelular compacta e organizada.

Larger Chondral Lesions Treated with Collagen Membrane - Matrix-Induced Autologous Chondrogenesis - Show Larger Increase in Clinical Scores.

Objective To evaluate clinically and radiologically the results of the treatment of chondral lesions using collagen membrane - autologous matrix-induced chondrogenesis (AMIC). Methods This is a series of observational cases, in which 15 patients undergoing AMIC were analyzed. The clinical evaluation was made by comparing the Lysholm and International Knee Document Commitee (IKDC) scores in the pre- and postoperative period of 12 months, and radiological evaluation using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score in the same postoperative period. Results The mean age of the patients was 39.2 years old, and the mean size of the chondral lesions was 1.55cm 2 . There was a significant improvement in clinical scores, with a mean increase of 24.6 points on Lysholm and of 24.3 on IKDC after 12 months. In the radiological evaluation, MOCART had a mean of 65 points. It was observed that the larger the size of the lesion, the greater the improvement in scores. Conclusion Evaluating subjective clinical scores, the treatment of chondral lesions with the collagen membrane showed good results, as well as the evaluation of MOCART, with greater benefit in larger lesions.

Larger Chondral Lesions Treated with Collagen Membrane - Matrix-Induced Autologous Chondrogenesis - Show Larger Increase in Clinical Scores / Lesões condrais maiores tratadas com uso de membrana de colágeno - condrogênese autóloga induzida por matriz - apresentam maior aumento nos escores clínicos

Abstract Objective To evaluate clinically and radiologically the results of the treatment of chondral lesions using collagen membrane - autologous matrix-induced chondrogenesis (AMIC). Methods This is a series of observational cases, in which 15 patients undergoing AMIC were analyzed. The clinical evaluation was made by comparing the Lysholm and International Knee Document Commitee (IKDC) scores in the pre- and postoperative period of 12 months, and radiological evaluation using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score in the same postoperative period. Results The mean age of the patients was 39.2 years old, and the mean size of the chondral lesions was 1.55cm2. There was a significant improvement in clinical scores, with a mean increase of 24.6 points on Lysholm and of 24.3 on IKDC after 12 months. In the radiological evaluation, MOCART had a mean of 65 points. It was observed that the larger the size of the lesion, the greater the improvement in scores. Conclusion Evaluating subjective clinical scores, the treatment of chondral lesions with the collagen membrane showed good results, as well as the evaluation of MOCART, with greater benefit in larger lesions.

Resumo Objetivo Avaliar clínica e radiologicamente os resultados do tratamento das lesões condrais com a membrana de colágeno - condrogênese autóloga induzida por matriz. Métodos Trata-se de uma série de casos observacional, na qual foram analisados 15 pacientes submetidos a condrogênese autóloga induzida por matriz. A avaliação clínica foi feita comparando os escores de Lysholm e International Knee Document Commitee (IKDC, na sigla em inglês) no pré- e pós-operatório de 12 meses, e avaliação radiológica através do escore de Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART, na sigla em inglês) no mesmo período de pós-operatório. Resultados A média de idade dos pacientes foi 39,2 anos, e a média do tamanho das lesões condrais foi de 1,55cm2. Houve uma melhora significativa nos escores clínicos, com média de aumento de 24,6 pontos no Lysholm e de 24,3 no IKDC, após 12 meses. Na avaliação radiológica, o MOCART teve média de 65 pontos. Observou-se que quanto maior o tamanho da lesão, maior foi a melhora nos escores. Conclusão Avaliando escores clínicos subjetivos, o tratamento das lesões condrais com a membrana de colágeno mostrou bons resultados, assim como a avaliação de MOCART, com maior benefício em lesões maiores.

WNT5A-Ca2+-CaN-NFAT signalling plays a permissive role during cartilage differentiation in embryonic chick digit development.

During digit development, the correct balance of chondrogenic signals ensures the recruitment of undifferentiated cells into the cartilage lineage or the maintenance of cells at the undifferentiated stage. WNT/ß catenin maintains the pool of progenitor cells, whereas TGFß signalling promotes cartilage differentiation by inducing Sox9 expression. Moreover, WNT5A promotes the degradation of ß catenin during mouse limb development. Although these mechanisms are well established, it is still unknown whether the signalling pathway downstream WNT5A is also involved in early chondrogenesis during digit formation. Thus, the aim of this study was to determine the role of WNT5A during the recruitment of progenitor cells during digit development. Our results showed that WNT5A activated calcium (Ca2+) release in the undifferentiated region during digit development. Further, the blockade of Ca2+ release or calcineurin (CaN) or nuclear factor of activated T-cells (NFAT) functions resulted in an inhibition of cartilage differentiation. Together, our results demonstrate that non canonical WNT5A-Ca2+-CaN-NFAT signalling plays a key role during embryonic digit development in vivo promoting the competence for chondrogenic signals and also acts as a permissive factor for chondrogenesis independently of cell death mechanisms.

Complete mid-portion rupture of the rat achilles tendon leads to remote and time-mismatched changes in uninjured regions.

PURPOSE: To examine healing adaptations over 17 weeks post Achilles tendon (AT) rupture in the injured region (IR) compared to an uninjured region (UIR) of the AT. METHODS: Twenty-four rats were subjected to a complete right-sided AT rupture, while the left side served as a control. ATs were harvested at 1, 2, 8 and 17 weeks post-rupture and stained with antibodies specific to Collagen type I (Col I) and II (Col II) as well as Alcian Blue and Picrosirius Red staining techniques. Histopathological changes, proteoglycan content, collagen alignment and immunoexpression were assessed. RESULTS: Both regions examined, IR and UIR, exhibited over weeks 1-17 similar healing adaptations of increasing collagen alignment, decreasing Col I immunoexpression, as well as increasing proteoglycan content and Col II occurrence. Increased proteoglycan content was found already at week 2 in the UIR, while it first increased at week 8 in the IR. The area positive to Col II was increased compared to controls at week 8 in the UIR, whereas it first raised at week 17 in the IR. Collagen disorganization successively declined to reach control levels at week 17 in the UIR, but was still higher in the IR. CONCLUSION: This study demonstrated that uninjured areas of the AT remote from the rupture site also undergo pronounced remodeling, although with time-span differences relative to injured AT portions. These changes including the pathologic heterotopic mineralization and chondrogenic differentiation observed in both regions may have implications in the choice of rehabilitation regimes in order to prevent secondary rupture.

Adipogenesis, Osteogenesis, and Chondrogenesis of Human Mesenchymal Stem/Stromal Cells: A Comparative Transcriptome Approach.

Adipogenesis, osteogenesis and chondrogenesis of human mesenchymal stem/stromal cells (MSC) are complex and highly regulated processes. Over the years, several studies have focused on understanding the mechanisms involved in the MSC commitment to the osteogenic, adipogenic and/or chondrogenic phenotypes. High-throughput methodologies have been used to investigate the gene expression profile during differentiation. Association of data analysis of mRNAs, microRNAs, circular RNAs and long non-coding RNAs, obtained at different time points over these processes, are important to depict the complexity of differentiation. This review will discuss the results that were highlighted in transcriptome analyses of MSC undergoing adipogenic, osteogenic and chondrogenic differentiation. The focus is to shed light on key molecules, main signaling pathways and biological processes related to different time points of adipogenesis, osteogenesis and chondrogenesis.

A human preadipocyte cell strain with multipotent differentiation capability as an in vitro model for adipogenesis.

Murine 3T3 cell lines constitute a standard model system for in vitro study of mammalian adipogenesis although they do not faithfully reflect the biology of the human adipose cells. Several human adipose cell lines and strains have been used to recapitulate human adipogenesis in vitro, but to date there is no generally accepted in vitro model for human adipogenesis. We obtained a clonal strain of human subcutaneous adipose stromal cells, IPI-SA3-C4, and characterized its utility as an in vitro model for human subcutaneous adipogenesis. IPI-SA3-C4 cells showed a high proliferative potential for at least 30 serial passages, reached 70 cumulative population doublings and exhibited a population doubling time of 47 h and colony forming efficiency of 12% at the 57th cumulative population doublings. IPI-SA3-C4 cells remained diploid (46XY) even at the 56th cumulative population doublings and expressed the pluripotency markers POU5F1, NANOG, KLF4, and MYC even at 50th cumulative population doublings. Under specific culture conditions, IPI-SA3-C4 cells displayed cellular hallmarks and molecular markers of adipogenic, osteogenic, and chondrogenic lineages and showed adipogenic capacity even at the 66th cumulative population doublings. These characteristics show IPI-SA3-C4 cells as a promising potential model for human subcutaneous adipogenesis in vitro.

Chondroinductive Alginate-Based Hydrogels Having Graphene Oxide for 3D Printed Scaffold Fabrication.

Scaffolds based on bioconjugated hydrogels are attractive for tissue engineering because they can partly mimic human tissue characteristics. For example, they can further increase their bioactivity with cells. However, most of the hydrogels present problems related to their processability, consequently limiting their use in 3D printing to produce tailor-made scaffolds. The goal of this work is to develop bioconjugated hydrogel nanocomposite inks for 3D printed scaffold fabrication through a micro-extrusion process having improved both biocompatibility and processability. The hydrogel is based on a photocrosslinkable alginate bioconjugated with both gelatin and chondroitin sulfate in order to mimic the cartilage extracellular matrix, while the nanofiller is based on graphene oxide to enhance the printability and cell proliferation. Our results show that the incorporation of graphene oxide into the hydrogel inks considerably improved the shape fidelity and resolution of 3D printed scaffolds because of a faster viscosity recovery post extrusion of the ink. Moreover, the nanocomposite inks produce anisotropic threads after the 3D printing process because of the templating of the graphene oxide liquid crystal. The in vitro proliferation assay of human adipose tissue-derived mesenchymal stem cells (hADMSCs) shows that bioconjugated scaffolds present higher cell proliferation than pure alginate, with the nanocomposites presenting the highest values at long times. Live/Dead assay otherwise displays full viability of hADMSCs adhered on the different scaffolds at day 7. Notably, the scaffolds produced with nanocomposite hydrogel inks were able to guide the cell proliferation following the direction of the 3D printed threads. In addition, the bioconjugated alginate hydrogel matrix induced chondrogenic differentiation without exogenous pro-chondrogenesis factors as concluded from immunostaining after 28 days of culture. This high cytocompatibility and chondroinductive effect toward hADMSCs, together with the improved printability and anisotropic structures, makes these nanocomposite hydrogel inks a promising candidate for cartilage tissue engineering based on 3D printing.

Understanding the Cellular and Molecular Mechanisms That Control Early Cell Fate Decisions During Appendicular Skeletogenesis.

The formation of the vertebrate skeleton is orchestrated in time and space by a number of gene regulatory networks that specify and position all skeletal tissues. During embryonic development, bones have two distinct origins: bone tissue differentiates directly from mesenchymal progenitors, whereas most long bones arise from cartilaginous templates through a process known as endochondral ossification. Before endochondral bone development takes place, chondrocytes form a cartilage analgen that will be sequentially segmented to form joints; thus, in the cartilage template, either the cartilage maturation programme or the joint formation programme is activated. Once the cartilage differentiation programme starts, the growth plate begins to form. In contrast, when the joint formation programme is activated, a capsule begins to form that contains special articular cartilage and synovium to generate a functional joint. In this review, we will discuss the mechanisms controlling the earliest molecular events that regulate cell fate during skeletogenesis in long bones. We will explore the initial processes that lead to the recruitment of mesenchymal stem/progenitor cells, the commitment of chondrocyte lineages, and the formation of skeletal elements during morphogenesis. Thereafter, we will review the process of joint specification and joint morphogenesis. We will discuss the links between transcription factor activity, cell-cell interactions, cell-extracellular matrix interactions, growth factor signalling, and other molecular interactions that control mesenchymal stem/progenitor cell fate during embryonic skeletogenesis.

Desarrollo y caracterización de películas de fibroina de seda para reparación condral / Development and characterization of silk fibroin films for condral reparation

RESUMEN La fibroína de seda es una proteína que ha demostrado ser un biomaterial con gran potencial en medicina regenerativa, por sus características de biocompatibilidad y su amplia posibilidad de modificación estructural permite ser usada como andamio favoreciendo procesos de crecimiento, diferenciación celular y la regeneración del tejido afectado. En este estudio se utilizaron capullos de gusano de seda Bombyx mori L., para la fabricación de películas de fibroína, los capullos fueron desgomados utilizando Na2CO3 0,02M, la fibroína obtenida se disolvió con LiBr 9,3M, el cual fue eliminado mediante diálisis y finalmente la solución de fibroína fue concentrada mediante contradiálisis. La fibroína fue servida en cajas de poliestireno, secadas a 90°C/24 horas y esterilizadas con etanol al 70%. Células madre mesenquimales fueron sembradas sobre estas películas de fibroína e inducidas a diferenciación utilizando un medio condrogénico especifico. La diferenciación fue evaluada por triplicado a los 14 y 21 días mediante extracción de ARN total, síntesis de ADN copia y amplificación por PCR de un grupo de genes específicos de cartílago empleando cebadores específicos. Se fabricaron películas de fibroína estables y resistentes que permitieron el crecimiento y la multiplicación celular, así como la diferenciación condrogénica evidenciada por la expresión de genes condrogenicos, no se afectó la viabilidad ni el recuento celular, las células interactuaron con el andamio evidenciado por el área de tapizado formado sobre la superficie de la película de fibroína. Finalmente se concluye que la fibroína de seda es un biomaterial que puede servir de andamio potencial para la regeneración de lesiones articulares.

ABSTRACT Silk fibroin is a protein that has been shown to be a biomaterial with great potential in regenerative medicine, due to its biocompatibility characteristics and its wide possibility of structural modification can be used as scaffold, favoring growth processes, cell differentiation and the regeneration of affected tissue. Bombix mori L. silkworm cocoons were used to make fibroin films, the silk fibroin were degummed using 0.02M Na2CO3, the obtained fibroin was dissolved with 9.3M LiBr, which was eliminated by dialysis and finally the fibroin solution was concentrated to 17% by counterdialysis. The fibroin was served in polystyrene boxes, dried at 90°C/24 hours and sterilized with 70% ethanol. The mesenchymal stem cells were seeded on fibroin films and induced differentiation using a specific chondrogenic medium. Differentiation was assessed in triplicate at 14 and 21 days by total RNA extraction, DNA synthesis copy and PCR amplification of a group of cartilage-specific genes using specific primers. Stable and resistant fibroin films that allowed cell growth and multiplication were fabricated, as well as the chondrogenic differentiation evidenced by the expression of chondrogenic genes, the viability and the cell count were not affected, the cells interacted with the scaffolding evidenced by the area of upholstery formed on the surface of the fibroin film. Finally, it is concluded that silk fibroin is a biomaterial that can serve as a potential scaffold for the regeneration of joint injuries.

Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect.

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106  cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.

Chondrogenic effect of liquid and gelled platelet lysate on canine adipose-derived mesenchymal stromal cells.

Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats / Efeito inibidor direto da cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos da cartilagem articular de ratos

The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)

O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)

Surgical treatment of chondral knee defects using a collagen membrane - autologus matrix-induced chondrogenesis / Tratamento cirúrgico das lesões condrais do joelho com o uso da membrana de colágeno - condrogênese autóloga induzida por matriz

ABSTRACT Objectives: To evaluate the clinical and functional results of patients diagnosed with full-thickness chondral defects on symptomatic knees who underwent a biological repair technique using autologous matrix-induced chondrogenesis. Methods: Seven patients who underwent surgical treatment due to chondral lesions in the knee by autologous matrix-induced chondrogenesis were evaluated. The Lysholm, Kujala and visual analog scale of pain questionnaires were applied before and 12 months after the surgery. Nuclear magnetic resonance images were evaluated 12 months after surgery according to MOCART (magnetic resonance observation of cartilage repair tissue) cartilage repair tissue score. Results: Of the seven patients evaluated, three presented defects classified as grade III and four as grade IV according to the International Cartilage Repair Society classification. Chondral defects were located in the medial femoral condyle (n = 2), patella (n = 2), and trochlea (n = 3). The mean age of the patients (six men and one woman) was 37.2 years (24-54 years). The mean chondral defect size was 2.11 cm2 (1.0-4.6 cm2). After 12 months, post-operative nuclear magnetic resonance showed resurfacing of the lesion site with scar tissue less thick than normal cartilage in all patients. The mean MOCART score was 66.42 points. A significant decrease in pain and an improvement in the Lysholm and Kujala scores were observed. Conclusion: The use of the collagen I/III porcine membrane was favorable for the treatment of chondral and osteochondral lesions of the knee when assessing the results using the VAS, Lysholm, and Kujala scores 1 year after surgery, as well as when assessing the magnetic resonance image of the lesion 6 months after surgery.

RESUMO Objetivos: Avaliar os resultados clínicos e funcionais dos pacientes com diagnóstico de lesões condrais de espessura total em joelhos sintomáticos submetidos a um método de reparação biológica por meio da técnica de condrogênese autóloga induzida por matriz. Métodos: Foram avaliados sete pacientes submetidos a tratamento cirúrgico devido a lesões condrais no joelho pela técnica de condrogênese autóloga induzida por matriz. Foram usados os questionários Lysholm e Kujala e a escala visual analógica da dor antes e após um ano de cirurgia. As imagens de ressonância nuclear magnética foram avaliadas após 12 meses de acordo com os critérios de reparo cartilaginoso de Mocart (magnetic resonance observation of cartilage repair tissue). Resultados: Dos sete pacientes avaliados, três apresentavam defeitos classificados como grau III e quatro como grau IV, de acordo com a classificação da International Cartilage Repair Society. Os defeitos condrais estavam no côndilo femoral medial (n = 2), na patela (n = 2) e na tróclea (n = 3). A média de idade dos sete pacientes (seis homens e uma mulher) foi de 37,2 anos (24 a 54). O tamanho médio dos defeitos condrais foi de 2,11 cm2 (1,0 a 4,6 cm2). Após 12 meses, a ressonância nuclear magnética pós-operatória mostrou preenchimento do local da lesão com tecido cicatricial menos espesso do que a cartilagem normal em todos os pacientes. O valor médio do questionário de Mocart após 12 meses foi de 66,42 pontos. Observou-se diminuição importante na dor e melhoria da avaliação dos questionários de Lysholm e Kujala. Conclusão: O uso da membrana de colágeno I/III de origem porcina se mostrou favorável no tratamento de lesões condrais e osteocondrais do joelho quando se avaliaram os resultados obtidos com a escala visual analógica da dor e o questionário de Lysholme Kujala um ano após a cirurgia, bem como quando se avaliou a imagem da lesão na ressonância magnética seis meses após a cirurgia.

Surgical treatment of chondral knee defects using a collagen membrane - autologus matrix-induced chondrogenesis.

OBJECTIVES: To evaluate the clinical and functional results of patients diagnosed with full-thickness chondral defects on symptomatic knees who underwent a biological repair technique using autologous matrix-induced chondrogenesis. METHODS: Seven patients who underwent surgical treatment due to chondral lesions in the knee by autologous matrix-induced chondrogenesis were evaluated. The Lysholm, Kujala and visual analog scale of pain questionnaires were applied before and 12 months after the surgery. Nuclear magnetic resonance images were evaluated 12 months after surgery according to MOCART (magnetic resonance observation of cartilage repair tissue) cartilage repair tissue score. RESULTS: Of the seven patients evaluated, three presented defects classified as grade III and four as grade IV according to the International Cartilage Repair Society classification. Chondral defects were located in the medial femoral condyle (n = 2), patella (n = 2), and trochlea (n = 3). The mean age of the patients (six men and one woman) was 37.2 years (24-54 years). The mean chondral defect size was 2.11 cm2 (1.0-4.6 cm2). After 12 months, post-operative nuclear magnetic resonance showed resurfacing of the lesion site with scar tissue less thick than normal cartilage in all patients. The mean MOCART score was 66.42 points. A significant decrease in pain and an improvement in the Lysholm and Kujala scores were observed. CONCLUSION: The use of the collagen I/III porcine membrane was favorable for the treatment of chondral and osteochondral lesions of the knee when assessing the results using the VAS, Lysholm, and Kujala scores 1 year after surgery, as well as when assessing the magnetic resonance image of the lesion 6 months after surgery.

OBJETIVOS: Avaliar os resultados clínicos e funcionais dos pacientes com diagnóstico de lesões condrais de espessura total em joelhos sintomáticos submetidos a um método de reparação biológica por meio da técnica de condrogênese autóloga induzida por matriz. MÉTODOS: Foram avaliados sete pacientes submetidos a tratamento cirúrgico devido a lesões condrais no joelho pela técnica de condrogênese autóloga induzida por matriz. Foram usados os questionários Lysholm e Kujala e a escala visual analógica da dor antes e após um ano de cirurgia. As imagens de ressonância nuclear magnética foram avaliadas após 12 meses de acordo com os critérios de reparo cartilaginoso de Mocart (magnetic resonance observation of cartilage repair tissue). RESULTADOS: Dos sete pacientes avaliados, três apresentavam defeitos classificados como grau III e quatro como grau IV, de acordo com a classificação da International Cartilage Repair Society. Os defeitos condrais estavam no côndilo femoral medial (n = 2), na patela (n = 2) e na tróclea (n = 3). A média de idade dos sete pacientes (seis homens e uma mulher) foi de 37,2 anos (24 a 54). O tamanho médio dos defeitos condrais foi de 2,11 cm2 (1,0 a 4,6 cm2). Após 12 meses, a ressonância nuclear magnética pós-operatória mostrou preenchimento do local da lesão com tecido cicatricial menos espesso do que a cartilagem normal em todos os pacientes. O valor médio do questionário de Mocart após 12 meses foi de 66,42 pontos. Observou-se diminuição importante na dor e melhoria da avaliação dos questionários de Lysholm e Kujala. CONCLUSÃO: O uso da membrana de colágeno I/III de origem porcina se mostrou favorável no tratamento de lesões condrais e osteocondrais do joelho quando se avaliaram os resultados obtidos com a escala visual analógica da dor e o questionário de Lysholme Kujala um ano após a cirurgia, bem como quando se avaliou a imagem da lesão na ressonância magnética seis meses após a cirurgia.