Bovine Cartilage Research Articles

Autoinduction-Based Quantification of In Situ TGF-β Activity in Native and Engineered Cartilage.

Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify in situ activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.

Synovial fluid does not retard fluid exudation during stress-relaxation of immature bovine cartilage

Interstitial fluid load support (FLS) is a dominant mechanism of lubrication in cartilage, producing a low friction coefficient while enhancing the tissue’s load bearing capabilities. Due to its viscosity, synovial fluid (SF) may retard loss of FLS by slowing the exudation of interstitial fluid from the cartilage. This study tested this hypothesis by comparing the stress-relaxation (SRL) response of immature bovine articular cartilage immersed either in phosphate buffered saline (PBS) or in healthy mature bovine SF, under unconfined compression (fluid exudation across cut lateral tissue boundary) and indentation testing (fluid exudation across articular surface). To investigate the influence of diffusion of SF molecular constituents into cartilage, the effect of incubation time in SF on SRL was also investigated. The SRL response in unconfined compression was not significantly different in PBS versus SF when compared directly (p = 0.98) and had a slope ofm = 1.00 ± 0.04 (R2 = 0.989 ± 0.007). Samples tested in PBS exhibited characteristic relaxation times, τPBS=42.6 ± 5.3 s andτSF = 40.8 ± 4.7 s, that were not significantly different (p = 0.40). Incubation time of 24 h in SF resulted in no significant difference in the SRL response (p = 0.39, m=1.03 ± 0.12; R2=0.983 ± 0.011, andτPBS = 43.4 ± 10.7 s versusτSF = 41.5 ± 4.8 s, p = 0.59). Indentation testing showed some statistically significant, but functionally insignificant, difference in SRL responses in PBS versus SF with a slope ofm = 0.958 ± 0.060 (R2 = 0.957 ± 0.020, p = 0.029, andτPBS = 16.9 ± 2.6 s versusτSF = 19.4 ± 3.3 s, p = 0.073). Based on these results, we reject the hypothesis that healthy SF can retard the loss of FLS in cartilage due to its viscosity.

Separation and purification of bovine nasal cartilage-derived chondroitin sulfate and evaluation of its binding to bovine serum albumin

This study employs an optimized and environmentally friendly method to extract and purify chondroitin sulfate (CS) from bovine nasal cartilage using enzymatic hydrolysis, ethanol precipitation, and DEAE Sepharose Fast Flow column chromatography. The extracted CS, representing 44.67 % ± 0.0016 of the cartilage, has a molecular weight of 7.62 kDa. Characterization through UV, FT-IR, NMR spectroscopy, and 2-aminoacridone derivatization HPLC revealed a high content of sulfated disaccharides, particularly ΔDi4S (73.59 %) and ΔDi6S (20.61 %). Interaction studies with bovine serum albumin (BSA) using fluorescence spectroscopy and molecular docking confirmed a high-affinity, static quenching interaction with a single binding site, primarily mediated by van der Waals forces and hydrogen bonding. The interaction did not significantly alter the polarity or hydrophobicity of BSA aromatic amino acids. These findings provide a strong foundation for exploring the application of CS in tissue engineering and drug delivery systems, leveraging its unique interaction with BSA for targeted delivery and enhanced efficacy.

Deciphering adiabatic rotating frame relaxometry in biological tissues.

This work aims to unravel the intricacies of adiabatic rotating frame relaxometry in biological tissues. The classical formalisms of dipolar relaxation and were systematically analyzed for water molecules reorienting on "fast" and "slow" timescales. These two timescales are, respectively, responsible for the absence and presence of dispersion. A time-averaged or over an adiabatic pulse duration was recast into a sum of and , but with different weightings. These weightings depend on the specific modulations of adiabatic pulse waveforms. In this context, stretched hyperbolic secant ( ) pulses were characterized. Previously published , continuous-wave (CW) , and measures from 12 agarose phantoms were used to validate the theoretical predictions. A similar validation was also performed on previously published ( =1, 4, 8) and from bovine cartilage specimens. Longitudinal relaxation weighting decreases for pulses as increases. Predicted CW values from agarose phantoms align well with the measured CW values, as indicated by a linear regression function: . The predicted adiabatic and from cartilage specimens are consistent with those previously measured, as quantified by: . This work has theoretically and experimentally demonstrated that adiabatic and can be recast into a sum of and , with varying weightings. Therefore, any suggestions that adiabatic rotating frame relaxometry in biological tissues could provide more information than the standard and warrant closer scrutiny.

A major functional role of synovial fluid is to reduce the rate of cartilage fatigue failure under cyclical compressive loading

ObjectiveBased on our recent study, which showed that cartilage fatigue failure in reciprocating sliding contact results from cyclical compressive forces, not from cyclical frictional forces, we hypothesize that a major functional role for synovial fluid (SF) is to reduce the rate of articular cartilage fatigue failure from cyclical compressive loading. DesignThe rate of cartilage fatigue failure due to repetitive compressive loading was measured by sliding a glass lens against an immature bovine cartilage tibial plateau strip immersed in mature bovine SF, phosphate-buffered saline (PBS), or SF/PBS dilutions (50% SF and 25% SF; n=8 for all four bath conditions). After 24 hours of reciprocating sliding (5,400 cycles), samples were visually assessed and if damage was observed, the test was terminated; otherwise, testing was continued for 72 hours (16,200 cycles), with solution refreshed daily. ResultsAll eight samples in the PBS group exhibited physical damage after 24 hours, with an average final surface roughness of Rq=0.210±0.067 mm. The SF group showed no damage after 24 hours; however, two of eight samples became damaged after 72 hours, producing a significantly lower average surface roughness than the PBS group (Rq=0.059±0.030 mm; p<10−4). For the remaining groups, at 72 hours one of eight samples damaged in the 50% SF group, and five of eight samples damaged in the 25% SF group. ConclusionsThe results strongly support our hypothesis, showing that decreased amounts of SF in the testing bath produces increased rates of fatigue failure in cartilage that was subjected to reciprocating sliding contact.

Extraction, Physicochemical Properties, and In Vitro Antioxidant Activities of Chondroitin Sulfate from Bovine Nose Cartilage.

Beef is an important high-nutrition livestock product, and several byproducts, such as bovine cartilage, are produced during slaughter. To effectively utilize these agricultural and pastoral byproducts, combined (trypsin-papain) enzymolysis and cetylpyridine chloride purification methods were used to obtain chondroitin sulfate (CS) from the nasal cartilage of Shaanxi Yellow cattle. The effects of pH, temperature, and time on the CS yield during enzymatic hydrolysis were investigated, and the CS extraction process was optimized using response surface methodology. The best yield of CS was 21.62% under the optimum conditions of pH 6.51, temperature of 64.53°C, and enzymolysis time of 19.86 h. The molecular weight of CS from Shaanxi cattle nasal cartilage was 89.21 kDa, glucuronic acid content was 31.76 ± 0.72%, protein content was 1.12 ± 0.03%, and sulfate group content was 23.34 ± 0.08%. The nasal cartilage CS of the Yellow cattle showed strong DPPH•, •OH, and ABTS+• radical scavenging abilities and ferrous reduction ability in the experimental concentration range. This study could contribute to "turn waste into treasure" and improve the comprehensive utilization of regional characteristic biological resources.

Cartilage Warping in Rhinoplasty: An Assessment of Temperature, Carving, and Suturing Conditions

Abstract Goals/Purpose Rhinoplasty is often contingent on the successful harvesting and allocation of cartilage in order to build the nasal framework. While autologous and cadaveric cartilage grafts have been utilized, it remains unclear how factors, such as suturing and carving techniques, as well as temperature, impact its short- and long-term warping potential. Our systematic review seeks to objectively assess how various carving (i.e. peripheral versus central, eccentric versus concentric), suture patterns (i.e. counter-balancing, oppositional), and temperature conditions impact the warping potential of cartilage grafts in rhinoplasty. In conducting this study, we hope to determine the optimal conditions for stable cartilage constructs in rhinoplasty. Methods/Technique A systematic literature review was conducted using a combination of terms including, “warp,” “rhinoplasty,” “cartilage,” “suture,” “temperature,” “carving,” and “cutting.” Removal of duplicates was performed, followed by further screening by abstract and full text. To reduce the possibility of bias, a standardized form was used for data extraction; variables including author, year of publication, journal of publication, study design, conditions, and results were recorded. Results/Complications A total of eleven studies were included in the analysis. Cartilage types included fresh frozen cadaveric graft, autologous costal cartilage, and bovine rib cartilage. Conditions associated with reduced warping include central and concentric based carving patterns, oppositional suturing; Peripherally and eccentric cut cartilage, as well as increased time, were associated with a higher degree of warping. Conclusion To the authors’ knowledge, our systematic analysis is the first to summarize the impact of various conditions on cartilage warping in rhinoplasty. Moreover, we determine the protective nature of certain carving and suturing methods - particularly central, concentric carving, and oppositional suturing - in minimizing cartilage warping. With this information, we hope to guide plastic surgeons in optimizing conditions to potentially prevent cartilage warping, and ultimately, provide reliable and sustainable results for patients undergoing rhinoplasty.

Zwitterionic polymer-dexamethasone conjugates penetrate and protect cartilage from inflammation

Improving the pharmacokinetics of intra-articularly injected therapeutics is a major challenge in treating joint disease. Small molecules and biologics are often cleared from the joint within hours, which greatly reduces their therapeutic efficacy. Furthermore, they are often injected at high doses, which can lead to local cytotoxicity and systemic side effects. In this study, we present modular polymer-drug conjugates of zwitterionic poly(carboxybetaine acrylamide) (pCBAA) and the anti-inflammatory glucocorticoid dexamethasone (DEX) to create cartilage-targeted carriers with slow-release kinetics. pCBAA polymers showed excellent cartilage penetration (full thickness in 1 h) and retention (>50 % after 2 weeks of washing). DEX was loaded onto the pCBAA polymer by employing two different DEX-bearing comonomers to produce pCBAA-co-DEX conjugates with different release kinetics. The slow-releasing conjugate showed zero-order release kinetics in PBS over 70 days. The conjugates elicited no oxidative stress on chondrocytes compared to dose-matched free DEX and protected bovine cartilage explants from the inflammatory response after treatment with IL-1β. By combining cartilage targeting and sustained drug release properties, the pCBAA-co-DEX conjugates solve many issues of today's intra-articular therapeutics, which could ultimately enable better long-term clinical outcomes with fewer side effects.

In Situ Loading and Time-Resolved Synchrotron-Based Phase Contrast Tomography for the Mechanical Investigation of Connective Knee Tissues: A Proof-of-Concept Study.

Articular cartilage and meniscus transfer and distribute mechanical loads in the knee joint. Degeneration of these connective tissues occurs during the progression of knee osteoarthritis, which affects their composition, microstructure, and mechanical properties. A deeper understanding of disease progression can be obtained by studying them simultaneously. Time-resolved synchrotron-based X-ray phase-contrast tomography (SR-PhC-µCT) allows to capture the tissue dynamics. This proof-of-concept study presents a rheometer setup for simultaneous in situ unconfined compression and SR-PhC-µCT of connective knee tissues. The microstructural response of bovine cartilage (n = 16) and meniscus (n = 4) samples under axial continuously increased strain, or two steps of 15% strain (stress-relaxation) is studied. The chondrocyte distribution in cartilage and the collagen fiber orientation in the meniscus are assessed. Variations in chondrocyte density reveal an increase in the top 40% of the sample during loading, compared to the lower half. Meniscus collagen fibers reorient perpendicular to the loading direction during compression and partially redisperse during relaxation. Radiation damage, image repeatability, and image quality assessments show little to no effects on the results. In conclusion, this approach is highly promising for future studies of human knee tissues to understand their microstructure, mechanical response, and progression in degenerative diseases.

Comparison of Minced Cartilage Implantation with Autologous Chondrocyte Transplantation in an In Vitro Inflammation Model.

The current gold standard to treat large cartilage defects is autologous chondrocyte transplantation (ACT). As a new surgical method of cartilage regeneration, minced cartilage implantation (MCI) is increasingly coming into focus. The aim of this study is to investigate the influence of chondrogenesis between isolated and cultured chondrocytes compared to cartilage chips in a standardized inflammation model with the proinflammatory cytokine TNFα. Articular chondrocytes from bovine cartilage were cultured according to the ACT method to passage 3 and transferred to spheroid culture. At the same time, cartilage was fragmented (<1 mm3) to produce cartilage chips. TNFα (20 ng/mL) was supplemented to simulate an inflammatory process. TNFα had a stronger influence on the passaged chondrocytes compared to the non-passaged ones, affecting gene expression profiles differently between isolated chondrocytes and cartilage chips. MCI showed less susceptibility to TNFα, with reduced IL-6 release and less impact on inflammation markers. Biochemical and histological analyses supported these findings, showing a greater negative influence of TNFα on the passaged pellet cultures compared to the unpassaged cells and MCI constructs. This study demonstrated the negative influence of TNFα on chondrogenesis in a chondrocyte spheroid culture and cartilage fragment model. Passaged chondrocytes are more sensitive to cytokine influences compared to non-passaged cells and chondrons. This suggests that MCI may have superior regeneration potential in osteoarthritic conditions compared to ACT. Further investigations are necessary for the translation of these findings into clinical practice.

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.

An update on a technical method of cartilage decellularization: (a physical-based protocol)

Introduction: Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods: The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results: The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusions: These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.

Возможности коррекции гормонального статуса, репродуктивной функции и нейровегетативных расстройств у пациенток с преждевременной недостаточностью яичников

Background: nowadays reproductive health condition tends to worsen. One of the problems is a decrease in the ovarian reserve, manifested in the form of premature ovarian insufficiency (POI), which initiates the infertility problem on the one hand, and the issue of premature menopause with all its accompanying socio-psychological aspects on the other Aim: to study the therapeutic possibilities of the bovine epiphyseal cartilage polypeptide in POI and anovulation in female patients aged 30–45 years old. Patients and Methods: the study included 40 female patients aged 30–45 years with a BMI of 18.5–29.9 kg/m2 and POI signs, determined by the following indicators: follicle-stimulating hormone (FSH) concentration ≥12 IU/L; anti-müllerian hormone (AMH) 0.2–1.2 ng/mL; antral follicle count (AFC) ≤5 during ultrasound on the 7th±2 day of the menstrual cycle; decreased serum estradiol concentration and anovulation lasting 1 to 5 years or with failed IVF. The female cohort was randomized into 2 groups of 20 persons: for two menstrual cycles, the main group received a drug for menopausal hormone therapy (MHT) containing 2 mg of estradiol and 10 mg of didrogesterone (per os 1 tablet daily in a cyclic regimen from day 1 to day 28) and the bovine epiphyseal cartilage polypeptide (lyophilizate for the preparation of a solution for intramuscular administration, 10 mg intramuscularly daily for 10 days from the 5th to the 14th day of the cycle). The control group received only MHT in the same regimen for two menstrual cycles. During the study, the changes of endometrial thickness and follicle growth, ovulation markers (corpus luteum), FSH, estradiol and AMH concentrations, the number of female patients with a normalized menstrual cycle and spontaneous conception were evaluated in both groups Results: in the main group there was a statistically significant (p&lt;0.05) decrease in FSH and luteinizing hormone (LH) levels relative to the baseline, an increase in estradiol concentration, an increase in the AFC and endometrial thickness, whereas the similar indicator changes were statistically insignificant in the control group. Besides, complete relief of neurovegetative symptoms was noted in the main group, and restoration of the ovulatory cycle was observed in 6 (30%) cases. There were no adverse events or complications during the drug regimen. Conclusion: the results obtained indicated the efficacy of the bovine epiphyseal cartilage polypeptide in the treatment of female patients with POI and anovulation and allowed to recommend the developed treatment regimen for practical use. KEYWORDS: premature ovarian insufficiency, follicle-stimulating hormone, anti-müllerian hormone, infertility, anovulation, menopausal disorders FOR CITATION: Khardikov A.V., Netyaga O.A. Correction of hormonal status, reproductive function and neurovegetative disorders in female patients with primary ovarian insufficiency. Russian Journal of Woman and Child Health. 2024;7(2):116–122 (in Russ.). DOI: 10.32364/2618- 8430-2024-7-2-5.

Evaluating the inhibition of IL-17A and TNFα in a cartilage explant model cultured with Th17-derived cytokines

IntroductionT-helper 17 (Th17) cells produce IL-17A playing a critical role in activating the pathogenic chain leading to joint tissue inflammation and destruction. Elevated levels of Th17 cells and IL-17A have been detected in skin lesions, blood, and synovial fluid from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (AS). Moreover, IL-17A inhibitors suppress disease activity in psoriasis, PsA and AS, supporting the evidence of IL-17A contributing to the disease pathogenesis. Although, IL-17A inhibitors are widely approved, it remains unclear how the inhibitory effect of IL-17A alters the extracellular matrix (ECM) of the joint in a Th17-conditioned inflammatory milieu. Therefore, the aim of this study was to establish a cartilage model cultured with conditioned medium from Th17 cells and inhibitors to explore the effect of IL-17A inhibition on joint tissue remodeling. MethodsNaïve CD4+ T cells from healthy human buffy coat were differentiated into Th17 cells, followed by Th17 cell activation to secrete Th17-related cytokines and molecules into media. The activated Th17 cells were isolated from the conditioned media (CM) and analyzed using flow cytometry to verify Th17 cell differentiation. The CM were assessed with ELISA to quantify the concentrations of cytokines secreted into the media by the Th17 cells. Healthy bovine cartilage explants were cultured with the Th17-CM and treated with IL-17A and TNFα inhibitors for 21 days. In harvested supernatant from the cartilage cultures, MMP- and ADAMTS-mediated biomarker fragments of type II collagen, aggrecan, and fibronectin were measured by ELISA to investigate the ECM remodeling within the cartilage tissue. ResultsTh17-CM stimulated a catabolic response in the cartilage. Markers of type II collagen and aggrecan degradation were upregulated, while anabolic marker of type II collagen formation remained on similar levels as the untreated explants. The addition of IL-17A inhibitor to Th17-CM decreased the elevated type II collagen and aggrecan degradation, however, degenerative levels were still elevated compared to untreated group. The addition of TNFα inhibitor completely reduced both type II collagen and aggrecan degradation compared to untreated explants. Moreover, the TNFα inhibitor treatment did not alter the type II collagen formation compared to untreated group. ConclusionThis study suggests that inhibition of IL-17A in Th17-conditioned cartilage tissue only partially reduced the MMP-mediated type II collagen degradation and ADAMTS-mediated aggrecan degradation, while the TNFα inhibitor treatment fully reduced both MMP- and ADAMTS-mediated ECM degradation. This exploratory study where ECM biomarkers are combined with Th17-conditioned ex vivo model may hold great potential as output for describing joint disease mechanisms and predicting structural effects of treatment on joint tissue.

CHONDROREGENERATIVE POTENTIAL ON SUBCUTANEOUS IMPLANTATION OF PLATELET-RICH FIBRIN (PRF)-IMPREGNATED DECELLULARIZED BOVINE CARTILAGE SCAFFOLD

The discovery of alternative implants with regenerative potential comparable to autologous cartilage continues to be encouraged because of the high donor site morbidity rate. This research tries to make an alternative implant that uses the concept of tissue engineering techniques in the form of endogenous regeneration by combining Decellularized Bovine Cartilage scaffold with Platelet-Rich Fibrin (BCPRF) which is implanted subcutaneously. The aim of this study was to compare the potential for chondroregeneration between BCPRF and autologous cartilage as assessed by chondrocyte cell formation, type 2 collagen thickness, and implant resorption rate in subcutaneous implantation. Using the research design method is a pretest-posttest control group design using New Zealand white rabbits. Forty eight experimental samples were divided into 2 groups which were treated with BCPRF and autologous cartilage implantation. Results were evaluated after 6 weeks. Evaluation was carried out on 39 samples. Microscopy showed better potential for autologous cartilage chondroregeneration than BCPRF with significant differences in the number of chondrocytes formed, the thickness of type 2 collagen (p=0.000), and the rate of implant resorption (p=0.000). In conclusion, the potential for chondroregeneration of autologous cartilage and BCPRF is significantly different in terms of the number of chondrocytes formed, the thickness of type 2 collagen, and the rate of implant resorption.

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.

Photo-Cross-Linkable, Injectable, and Highly Adhesive GelMA-Glycol Chitosan Hydrogels for Cartilage Repair.

Hydrogels provide a promising platform for cartilage repair and regeneration. Although hydrogels have shown some efficacy, they still have shortcomings including poor mechanical properties and suboptimal integration with surrounding cartilage. Herein, hydrogels that are injectable, cytocompatible, mechanically robust, and highly adhesive to cartilage are developed. This approach uses GelMA-glycol chitosan (GelMA-GC) that is crosslinkable with visible light and photoinitiators (lithium acylphosphinate and tris (2,2'-bipyridyl) dichlororuthenium (II) hexahydrate ([RuII(bpy)3 ]2+ and sodium persulfate (Ru/SPS)). Ru/SPS-cross-linked hydrogels have higher compressive and tensile modulus, and most prominently higher adhesive strength with cartilage, which also depends on inclusion of GC. Tensile and push-out tests of the Ru/SPS-cross-linked GelMA-GC hydrogels demonstrate adhesive strength of ≈100 and 46kPa, respectively. Hydrogel precursor solutions behave in a Newtonian manner and are injectable. After injection in focal bovine cartilage defects and in situ cross-linking, this hydrogel system remains intact and integrated with cartilage following joint manipulation ex vivo. Cells remain viable (>85%) in the hydrogel system and further show tissue regeneration potential after three weeks ofin vitroculture. These preliminary results provide further motivation for future research on bioadhesive hydrogels for cartilage repair and regeneration.

Ex Vivo Functional Benchmarking of Hyaluronan-Based Osteoarthritis Viscosupplement Products: Comprehensive Assessment of Rheological, Lubricative, Adhesive, and Stability Attributes.

While many injectable viscosupplementation products are available for osteoarthritis (OA) management, multiple hydrogel functional attributes may be further optimized for efficacy enhancement. The objective of this study was to functionally benchmark four commercially available hyaluronan-based viscosupplements (Ostenil, Ostenil Plus, Synvisc, and Innoryos), focusing on critical (rheological, lubricative, adhesive, and stability) attributes. Therefore, in vitro and ex vivo quantitative characterization panels (oscillatory rheology, rotational tribology, and texture analysis with bovine cartilage) were used for hydrogel product functional benchmarking, using equine synovial fluid as a biological control. Specifically, the retained experimental methodology enabled the authors to robustly assess and discuss various functional enhancement options for hyaluronan-based hydrogels (chemical cross-linking and addition of antioxidant stabilizing agents). The results showed that the Innoryos product, a niacinamide-augmented linear hyaluronan-based hydrogel, presented the best overall functional behavior in the retained experimental settings (high adhesivity and lubricity and substantial resistance to oxidative degradation). The Ostenil product was conversely shown to present less desirable functional properties for viscosupplementation compared to the other investigated products. Generally, this study confirmed the high importance of formulation development and control methodology optimization, aiming for the enhancement of novel OA-targeting product critical functional attributes and the probability of their clinical success. Overall, this work confirmed the tangible need for a comprehensive approach to hyaluronan-based viscosupplementation product functional benchmarking (product development and product selection by orthopedists) to maximize the chances of effective clinical OA management.

Sensitivity of simulated knee joint mechanics to selected human and bovine fibril-reinforced poroelastic material properties

Fibril-reinforced poroviscoelastic material models are considered state-of-the-art in modeling articular cartilage biomechanics. Yet, cartilage material parameters are often based on bovine tissue properties in computational knee joint models, although bovine properties are distinctly different from those of humans. Thus, we aimed to investigate how cartilage mechanical responses are affected in the knee joint model during walking when fibril-reinforced poroviscoelastic properties of cartilage are based on human data instead of bovine. We constructed a finite element knee joint model in which tibial and femoral cartilages were modeled as fibril-reinforced poroviscoelastic material using either human or bovine data. Joint loading was based on subject-specific gait data. The resulting mechanical responses of knee cartilage were compared between the knee joint models with human or bovine fibril-reinforced poroviscoelastic cartilage properties. Furthermore, we conducted a sensitivity analysis to determine which fibril-reinforced poroviscoelastic material parameters have the greatest impact on cartilage mechanical responses in the knee joint during walking. In general, bovine cartilage properties yielded greater maximum principal stresses and fluid pressures (both up to 30%) when compared to the human cartilage properties during the loading response in both femoral and tibial cartilage sites. Cartilage mechanical responses were very sensitive to the collagen fibril-related material parameter variations during walking while they were unresponsive to proteoglycan matrix or fluid flow-related material parameter variations. Taken together, human cartilage material properties should be accounted for when the goal is to compare absolute mechanical responses of knee joint cartilage as bovine material parameters lead to substantially different cartilage mechanical responses.

Preparation of low molecular weight chondroitin sulfate from different sources by H2O2/ascorbic acid degradation and its degradation mechanism

Low molecular weight chondroitin sulfate (LMCS) has attention for enhanced bioavailability and bioactivity compared to native CS. We optimized H2O2/ ascorbic acid (Vc) degradation conditions to prepare LMCS from chicken, bovine, and shark cartilages. Degradation kinetics models and chemical composition data of LMCS showed the GlcA residues of chondroitin-4-sulfate (CSA) may be preferentially attacked. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) indicated that the CH of GlcA in CS was broken through a hydrogen abstraction reaction to break the β-(1 → 3) bond and form the hexendioic acid product. Standard density functional theory (DFT) calculations indicated that the energy required for the hydrogen abstraction from the C1-H bond in GlcA was lower than that of GalNAc. Molecular dynamics (MD) showed that CSA was more likely to interact with hydroxyl radicals (·OH) than non-sulfated chondroitin (CSO) and chondroitin-6-sulfate (CSC). These results provide guidance for producing LMCS.