Multi-layered electrospinning and electrospraying approach: Effect of polymeric supplements on chondrocyte suspension

Articular cartilage was expected to be one of the first tissues to be successfully engineered, but replicating the complex fibril architecture and the cellular distribution of the native cartilage has proven difficult. While electrospinning has been widely used to reproduce the depth-dependent fibre architecture in 3D scaffolds, the chondrocyte-controlled distribution remains an unsolved problem. To incorporate cells homogeneously through the depth of scaffolds, a combination of polymer electrospinning and cell seeding is necessary. A multi-layer approach alternating between polymer electrospinning with chondrocyte electrospraying can be a solution. Still, the success of this process is related to the survival rate of the electrosprayed chondrocytes embedded within the electrospun mesh. In this regard, the present study investigated the impact of the multi-layered process and the supplementation of the electrospray chondrocyte suspension with different concentrations of Gelatin and Alginate on the viability of electrosprayed chondrocytes embedded within a Polycaprolactone/Gelatin electrospun mesh and on the mechanical properties of the resulting meshes. The addition of Gelatin in the chondrocyte suspension did not increase significantly ( p > 0.05) the percentage of viable electrosprayed chondrocytes (25%), while 3 wt% Alginate addition led to a significant ( p < 0.05) increase in chondrocyte viability (50%) relative to the case without polymer supplement (15%). Furthermore, the addition of both polymer supplements increased the mechanical properties of the multi-layer construct. These findings imply that this multi-layered approach can be applied to cartilage TE allowing for automated chondrocyte integration during scaffolds creation.

The Morphological, Clinical and Radiological Outputs of the Preclinical Study After Treatment of the Osteochondral Lesions in the Porcine Knee Model Using Implantation of Scaffold Based on the of Calcium Phosphate Biocement

The symptomatic full-thickness cartilage lesions or cartilage degeneration leads to the destruction of the normal chondral architecture and bone structure in affected area, causes the osteoarthritis, and general damage to the health. Knee joints are most frequently affected by this condition. The permanent damage of the articular cartilage and subchondral bone has motivated many scientists and clinicians to explore new methods of regeneration of osteochondral defects, such as novel materials. We studied the potential of the biocement based on calcium phosphate consisting of a mixture of four amino acids (glycine, proline, hydroxyproline and lysine) in the regenerating process of the artificially created osteochondral defect on the porcine medial femoral condyle in the stifle joint. The mass ratio of the amino acids in biocement CAL was 4:2:2:1. The Ca/P ratio in cement was 1.67 which correspond with ratio in hydroxyapatite. We compared the results with spontaneous healing of an artificially created cyst with that of the healthy tissue. The animal group treated with biocement paste CAL presented completely filled osteochondral defects. The results were confirmed by histological and radiological assessments, which have shown regenerated chondral and bone tissue in the examined knee joints. Macroscopic evaluation showed that neocartilage was well integrated with the adjacent native cartilage in animal group with biocement CAL, compared with healing of the artificial cyst, where treated cartilage surfaces were visibly lower than the surrounding native cartilage surface and a border between native and restored tissue was apparent. The qualitative assessment of the implant histology specimens showed full regeneration of the hyaline cartilage and subchondral bone in animals with biocement CAL. The artificial cyst group showed remarkable fibrillation. The detailed MRI analysis of cross-section of osteochondral defect confirmed the complete cartilage and subchondral bone healing where the thickness of the regenerated cartilage was 1.5 mm. The MRI imaging of defects in the artificial cyst group showed incomplete healing, neo cartilage tissue reduced up to 50%.

Resolving the Near-Infrared Spectrum of Articular Cartilage

Objective Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by ( a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and ( b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. Design The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples. Results As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models. Conclusion Spectral peaks in the regions 5500 to 6250 cm−1 and 8100 to 8600 cm−1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

Is T2 mapping reliable in evaluation of native and repair cartilage tissue of the knee?

Purpose To evaluate the effect of imaging plane and experience of observers on the reliability of T2 mapping of native and repair cartilage tissue of the knee. Methods Fifteen consecutive patients from two randomised controlled trials (RCTs) were included in this cross-sectional study. Patients with an isolated knee cartilage lesion were randomised to receive either debridement or microfracture (RCT 1) or debridement or autologous chondrocyte implantation (RCT 2). T2 mapping was performed in coronal and sagittal planes two years postoperatively. A musculoskeletal radiologist, a resident of radiology and two orthopaedic surgeons measured the T2 values independently. Intraclass Correlation Coefficient (ICC) with 95% Confidence Intervals was used to calculate the inter- and intraobserver agreement. Results Mean age for the patients was 36.8 ± 11 years, 8 (53%) were men. The overall interobserver agreement varied from poor to good with ICCs in the range of 0.27– 0.76 for native cartilage and 0.00 – 0.90 for repair tissue. The lowest agreement was achieved for evaluations of repair cartilage tissue. The estimated ICCs suggested higher inter- and intraobserver agreement for radiologists. On medial femoral condyles, T2 values were higher for native cartilage on coronal images (p < 0.001) and for repair tissue on sagittal images (p < 0.001). Conclusions The reliability of T2 mapping of articular cartilage is influenced by the imaging plane and the experience of the observers. This influence may be more profound for repair cartilage tissue. This is important to consider when using T2 mapping to measure outcomes after cartilage repair surgery. Trial registration ClinicalTrials.gov, NCT02637505 and NCT02636881, registered December 2015. Level of evidence II, based on prospective data from two RCTs.

Micropartículas de PLLA y CHT como andamiaje para la regeneración del cartílago articular: Modelo animal.

The avascular nature of cartilaginous tissue has historically lead to bad prognosis in osteochondral injuries. One of the possible treatment options of these injuries is the use of scaffolds, being superior to other options that obtain a fibrous cartilage as a result. We have elaborated PLLA and CHT microspheres as a scaffold for the treatment of osteochondral injuries carried out in albine New Zealand rabbits. They were distributed into groups with different proportions of microspheres, having also a control group with untreated injuries. Native cartilage of the contralateral knees was also analysed. Samples were evaluated in order to establish the quality of the cartilage obtained (using de macroscopic ICRS, microscopic ICRS II scales and a histomorphometric study).The groups with microspheres obtained a regeneration cartilage with hyaline characteristics, a good cell distribution and regular surface. The control group resulted in a cartilage with worse organization and an irregular surface.

In Vitro Evaluation of the Influence of Substrate Mechanics on Matrix-Assisted Human Chondrocyte Transplantation

Matrix-assisted chondrocyte transplantation (MACT) is of great interest for the treatment of patients with cartilage lesions. However, the roles of the matrix properties in modulating cartilage tissue integration during MACT recovery have not been fully understood. The objective of this study was to uncover the effects of substrate mechanics on the integration of implanted chondrocyte-laden hydrogels with native cartilage tissues. To this end, agarose hydrogels with Young’s moduli ranging from 0.49 kPa (0.5%, w/v) to 23.08 kPa (10%) were prepared and incorporated into an in vitro human cartilage explant model. The hydrogel-cartilage composites were cultivated for up to 12 weeks and harvested for evaluation via scanning electron microscopy, histology, and a push-through test. Our results demonstrated that integration strength at the hydrogel-cartilage interface in the 1.0% (0.93 kPa) and 2.5% (3.30 kPa) agarose groups significantly increased over time, whereas hydrogels with higher stiffness (>8.78 kPa) led to poor integration with articular cartilage. Extensive sprouting of extracellular matrix in the interfacial regions was only observed in the 0.5% to 2.5% agarose groups. Collectively, our findings suggest that while neocartilage development and its integration with native cartilage are modulated by substrate elasticity, an optimal Young’s modulus (3.30 kPa) possessed by agarose hydrogels is identified such that superior quality of tissue integration is achieved without compromising tissue properties of implanted constructs.