Thermoreversible hydrogel scaffolds for articular cartilage engineering

Articular cartilage has limited potential for repair. Current clinical treatments for articular cartilage damage often result in fibrocartilage and are associated with joint pain and stiffness. To address these concerns, researchers have turned to the engineering of cartilage grafts. Tissue engineering, an emerging field for the functional restoration of articular cartilage and other tissues, is based on the utilization of morphogens, scaffolds, and responding progenitor/stem cells. Because articular cartilage is a water-laden tissue and contains within its matrix hydrophilic proteoglycans, an engineered cartilage graft may be based on synthetic hydrogels to mimic these properties. To this end, we have developed a polymer system based on the hydrophilic copolymer poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)]. Solutions of this polymer are liquid below 25 degrees C and gel above 35 degrees C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. The objective of this work was to determine the effects of the hydrogel components on the phenotype of encapsulated chondrocytes. Bovine articular chondrocytes were used as an experimental model. Results demonstrated that the components required for hydrogel fabrication did not significantly reduce the proteoglycan synthesis of chondrocytes, a phenotypic marker of chondrocyte function. In addition, chondrocyte viability, proteoglycan synthesis, and type II collagen synthesis within P(PF-co-EG) hydrogels were investigated. The addition of bone morphogenetic protein-7 increased chondrocyte proliferation with the P(PF-co-EG) hydrogels, but did not increase proteoglycan synthesis by the chondrocytes. These results indicate that the temperature-responsive P(PF-co-EG) hydrogels are suitable for chondrocyte delivery for articular cartilage repair.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

Bioactive Stratified Polymer Ceramic-Hydrogel Scaffold for Integrative Osteochondral Repair

Due to the intrinsically poor repair potential of articular cartilage, injuries to this soft tissue do not heal and require clinical intervention. Tissue engineered osteochondral grafts offer a promising alternative for cartilage repair. The functionality and integration potential of these grafts can be further improved by the regeneration of a stable calcified cartilage interface. This study focuses on the design and optimization of a stratified osteochondral graft with biomimetic multi-tissue regions, including a pre-designed and pre-integrated interface region. Specifically, the scaffold based on agarose hydrogel and composite microspheres of polylactide-co-glycolide (PLGA) and 45S5 bioactive glass (BG) was fabricated and optimized for chondrocyte density and microsphere composition. It was observed that the stratified scaffold supported the region-specific co-culture of chondrocytes and osteoblasts which can lead to the production of three distinct yet continuous regions of cartilage, calcified cartilage and bone-like matrices. Moreover, higher cell density enhanced chondrogenesis and improved graft mechanical property over time. The PLGA-BG phase promoted chondrocyte mineralization potential and is required for the formation of a calcified interface and bone regions on the osteochondral graft. These results demonstrate the potential of the stratified scaffold for integrative cartilage repair and future studies will focus on scaffold optimization and in vivo evaluations.     

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Thermosensitive chitosan–Pluronic hydrogel as an injectable cell delivery carrier for cartilage regeneration

Injectable hydrogels have been studied for potential applications for articular cartilage regeneration. In this study, a thermosensitive chitosan–Pluronic (CP) hydrogel was designed as an injectable cell delivery carrier for cartilage regeneration. The CP conjugate was synthesized by grafting Pluronic onto chitosan using EDC/NHS chemistry. The sol–gel phase transition and mechanical properties of the CP hydrogel were examined by rheological experiments. The CP solution underwent a sol–gel transition around 25 °C at which the storage modulus (G′) approaches 10⁴ Pa, highlighting the potential of this material as an injectable scaffold for cartilage regeneration. The CP hydrogel was formed rapidly by increasing the temperature. The morphology of the dried CP hydrogel was observed by scanning electron microscopy. In vitro cell culture was performed using bovine chondrocytes. The proliferation of bovine chondrocytes and the amount of synthesized glycosaminoglycan increased for 28 days. These results suggested that the CP hydrogel has potential as an injectable cell delivery carrier for cartilage regeneration and could serve as a new biomaterial for tissue engineering.     

Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering

Injectable cartilaginous constructs that can form gels in tissue defects have many advantages in tissue engineering applications. In this study we created an injectable hydrogel consisting of methacrylated glycol chitosan (MeGC) and hyaluronic acid (HA) by photocrosslinking with a riboflavin photoinitiator under visible light. A minimum irradiation time of 40 s was required to produce stable gels for cell encapsulation with 87–90% encapsulated chondrocyte viability. Although increasing the irradiation time from 40 to 600 s significantly enhanced the compressive modulus of the hydrogels up to 11 or 17 kPa for MeGC or MeGC/HA, respectively, these conditions reduced the encapsulated cell viability to 60–65%. The majority of chondrocytes encapsulated in MeGC hydrogels after 300 s irradiation maintained a rounded shape with a high cell viability of ～80–87% over a 21 day culture period. The incorporation of HA in MeGC hydrogels increased the proliferation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. These findings demonstrate that MeGC/HA composite hydrogels have the potential for cartilage repair.     

Introducing chemical functionality in Fmoc-peptide gels for cell culture

Aromatic short peptide derivatives, i.e. peptides modified with aromatic groups such as 9-fluorenylmethoxycarbonyl (Fmoc), can self-assemble into self-supporting hydrogels. These hydrogels have some similarities to extracellular matrices due to their high hydration, relative stiffness and nanofibrous architecture. We previously demonstrated that Fmoc-diphenylalanine (Fmoc-F₂) provides a suitable matrix for two-dimensional (2D) or three-dimensional (3D) culture of primary bovine chondrocytes. In this paper we investigate whether the introduction of chemical functionality, such as NH₂, COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F₂ and n-protected Fmoc amino acids, lysine (K, with side chain R = (CH₂)₄NH₂), glutamic acid (D, with side chain R = CH₂COOH), and serine (S, with side chain R = CH₂OH) were studied. All compositions produced fibrous scaffolds with fibre diameters in the range of 32–65 nm as assessed by cryo-scanning electron microscopy and atomic force microscopy. Fourier transform infrared spectroscopy analysis suggested that peptide segments adopt a predominantly antiparallel β-sheet conformation. Oscillatory rheology results show that all four hydrogels have mechanical profiles of soft viscoelastic materials with elastic moduli dependent on the chemical composition, ranging from 502 Pa (Fmoc-F₂/D) to 21.2 KPa (Fmoc-F₂). All gels supported the viability of bovine chondrocytes as assessed by a live–dead staining assay. Fmoc-F₂/S and Fmoc-F₂/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F₂/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F₂/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F₂/S gel was shown to support retention of cell morphology in 3D culture of bovine chondrocytes. These results demonstrate that introduction of chemical functionality into Fmoc-peptide scaffolds may provide gels with tunable chemical and mechanical properties for in vitro cell culture.     

Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering

Glycopolypeptides are an emerging class of bioinspired polymers that mimic naturally occurring glycopeptides or glycoproteins, and therefore are expected to exhibit great potential for biomedical applications. In this study, a glycopolypeptide was synthesized by conjugation of poly(γ-propargyl-l-glutamate) (PPLG) with azido-modified mannose and 3-(4-hydroxyphenyl) propanamide (HPPA), via click chemistry. Injectable hydrogels based on the glycopolypeptide were developed through enzymatic crosslinking reaction in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The physicochemical properties of the hydrogels, such as gelation time, storage modulus, swelling and degradation time, could be controlled by varying the concentrations of HRP and H₂O₂. The glycopolypetide copolymer as well as the extracts of the glycopolypetide hydrogels displayed good cytocompatibility in vitro. After subcutaneous injection into rats, the glycopolypeptide hydrogels were rapidly formed in situ, and exhibited acceptable biocompatibility accompanying the degradation of the hydrogels in vivo. The rabbit chondrocytes inside the glycopolypeptide hydrogels showed spherical morphology with high viability during the incubation period of 3 weeks in vitro, and exhibited a higher proliferation rate than within the hydrogel counterparts of PPLG grafted with 2-(2-(2-methoxyethoxy)ethoxy)ethane (MEO₃) and HPPA. Biochemical analysis demonstrated that the production of glycosaminoglycans (GAG) and type II collagen were significantly enhanced after incubation for 2 and 3 weeks in vitro. Moreover, the chondrocyte-containing glycopolypeptide hydrogels in subcutaneous model of nude mice maintained chondrocyte phenotype and produced the cartilaginous specific matrix. These results indicated that the biomimetic glycopolypeptide-based hydrogels hold potential as three-dimensional scaffolds for cartilage tissue engineering.     

The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures

Pulsed low intensity ultrasound (PLIUS) has been used successfully for bone fracture repair and has therefore been suggested for cartilage regeneration. However, previous in vitro studies with chondrocytes show conflicting results as to the effect of PLIUS on the elaboration of extracellular matrix. This study tests the hypothesis that PLIUS, applied for 20 min/day, stimulates the synthesis of sulphated glycosaminoglycan (sGAG) by adult bovine articular chondrocytes cultured in either monolayer or agarose constructs. For both culture models, PLIUS at either 30 or 100 mW/cm² intensity had no net effect on the total sGAG content. Although PLIUS at 100 mW/cm² did induce a 20% increase in sGAG content at day 2 of culture in agarose, this response was lost by day 5. Intensities of 200 and 300 mW/cm² resulted in cell death probably due to heating from the ultrasound transducers. The lack of a sustained up-regulation of sGAG synthesis may reflect the suggestion that PLIUS only induces a stimulatory effect in the presence of a tissue injury response. These results suggest that PLIUS has a limited potential to provide an effective method of stimulating matrix production as part of a tissue engineering strategy for cartilage repair.     

Primary human chondrocyte extracellular matrix formation and phenotype maintenance using RGD-derivatized PEGDM hydrogels possessing a continuous Young’s modulus gradient

Efficient ex vivo methods for expanding primary human chondrocytes while maintaining the phenotype is critical to advancing the sourcing of autologous cells for tissue engineering applications. While there has been significant research reported in the literature, systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using limited cell numbers. Functionalized hydrogels possessing continuous variations in physico-chemical properties are, therefore, an efficient three-dimensional platform for studying several properties simultaneously. Herein we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system with a modulus gradient (～27,000–3800 Pa) containing a uniform concentration of arginine–glycine–aspartic acid (RGD) peptide to enhance cell adhesion in order to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance, and extracellular matrix (ECM) production with hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with a higher storage modulus (>13,100 Pa), while regions with a lower storage modulus maintained their cell number and phenotype. Over 3 weeks culture hydrogel regions possessing a lower Young's modulus experienced an increase in ECM content (～200%) compared with regions with a higher storage modulus. Variations in the amount and organization of the cytoskeletal markers actin and vinculin were observed within the modulus gradient, which are indicative of differences in chondrogenic phenotype maintenance and ECM expression. Thus scaffold mechanical properties have a significant impact in modulating human osteoarthritic chondrocyte behavior and tissue formation.     

β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker.     

The clustering and morphology of chondrocytes in normal and mildly degenerate human femoral head cartilage studied by confocal laser scanning microscopy

Chondrocytes are the major cell type present in hyaline cartilage and they play a crucial role in maintaining the mechanical resilience of the tissue through a balance of the synthesis and breakdown of extracellular matrix macromolecules. Histological assessment of cartilage suggests that articular chondrocytes in situ typically occur singly and demonstrate a rounded/elliptical morphology. However, there are suggestions that their grouping and fine shape is more complex and that these change with cartilage degeneration as occurs in osteoarthritis. In the present study we have used confocal laser scanning microscopy and fluorescently labelled in situ human chondrocytes and advanced imaging software to visualise chondrocyte clustering and detailed morphology within grade‐0 (non‐degenerate) and grade‐1 (mildly degenerate) cartilage from human femoral heads. Graded human cartilage explants were incubated with 5‐chloromethylfluorescein diacetate and propidium iodide to identify the morphology and viability, respectively, of in situ chondrocytes within superficial, mid‐ and deep zones. In grade‐0 cartilage, the analysis of confocal microscope images showed that although the majority of chondrocytes were single and morphologically normal, clusters (i.e. three or more chondrocytes within the enclosed lacunar space) were occasionally observed in the superficial zone, and 15–25% of the cell population exhibited at least one cytoplasmic process of ~ 5 μm in length. With degeneration, cluster number increased (~ 50%) but not significantly; however, the number of cells/cluster (P < 0.001) and the percentage of cells forming clusters increased (P = 0.0013). In the superficial zone but not the mid‐ or deep zones, the volume of clusters and average volume of chondrocytes in clusters increased (P < 0.001 and P < 0.05, respectively). The percentage of chondrocytes with processes, the number of processes/cell and the length of processes/cell increased in the superficial zone of grade‐1 cartilage (P = 0.0098, P = 0.02 and P < 0.001, respectively). Processes were categorised based on length (L0 – no cytoplasmic processes; L1 < 5 μm; 5 < L2 ≤ 10 μm; 10 < L3 ≤ 15 μm; L4 > 15 μm). With cartilage degeneration, for chondrocytes in all zones, there was a significant decrease (P = 0.015) in the percentage of chondrocytes with ‘normal’ morphology (i.e. L0), with no change in the percentage of cells with L1 processes; however, there were significant increases in the other categories. In grade‐0 cartilage, chondrocyte clustering and morphological abnormalities occurred and with degeneration these were exacerbated, particularly in the superficial zone. Chondrocyte clustering and abnormal morphology are associated with aberrant matrix metabolism, suggesting that these early changes to chondrocyte properties may be associated with cartilage degeneration.     

The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects

Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote the chondrogenesis, as suggested by the detection of TGF-β1 in both in vitro and in vivo cell/hydrogel constructs.     

Unconfined compression properties of a porous poly(vinyl alcohol)–chitosan-based hydrogel after hydration

A poly(vinyl alcohol) (PVA) hydrogel composite scaffold containing N,O-carboxymethylated chitosan (NOCC) was tested to assess its potential as a scaffold for cartilage tissue engineering in a weight-bearing environment. The mechanical properties under unconfined compression for different hydration periods were investigated. The effect of supplementing PVA with NOCC (20 wt.% PVA:5 vol.% NOCC) produced a porosity of 43.3% and this was compared against a non-porous PVA hydrogel (20 g PVA: 100 ml of water, control). Under non-hydrated conditions, the porous PVA–NOCC hydrogel behaved in a similar way to the control non-porous PVA hydrogel, with similar non-linear stress–strain response under unconfined compression (0–30% strain). After 7 days’ hydration, the porous hydrogel demonstrated a reduced stiffness (0.002 kPa, at 25% strain), resulting in a more linear stiffness relationship over a range of 0–30% strain. Poisson’s ratio for the hydrated non-porous and porous hydrogels ranged between 0.73 and 1.18, and 0.76 and 1.33, respectively, suggesting a greater fluid flow when loaded. The stress relaxation function for the porous hydrogel was affected by the hydration period (from 0 to 600 s); however the percentage stress relaxation regained by about 95%, after 1200 s for all hydration periods assessed. No significant differences were found between the different hydration periods between the porous hydrogels and control. The calculated aggregate modulus, H_A, for the porous hydrogel reduced drastically from 10.99 kPa in its non-hydrated state to about 0.001 kPa after 7 days’ hydration, with the calculated shear modulus reducing from 30.92 to 0.14 kPa, respectively. The porous PVA–NOCC hydrogel conformed to a biphasic, viscoelastic model, which has the desired properties required for any scaffold in cartilage tissue engineering.     

Minocycline reduces articular cartilage damage following osteochondral injury

IntroductionSecondary injury pathways activated after chondral and osteochondral injury represent a potential target for therapies designed to minimize articular cartilage loss. The primary objective of this study was to test the potential chondroprotective effects of intra-articular minocycline following osteochondral injury.MethodsIn vitro experiments were first performed with rabbit femoral condyles explants using an osteochondral drill injury model. Data from these in vitro experiments showed that minocycline at concentrations of 10–1000 nM decreased chondrocyte apoptosis in a dose-dependent manner. In vivo experiments were then conducted using the same injury model, studying the effects of intra-articular minocycline on chondrocyte apoptosis, chondrocyte cell number, and cartilage thickness.ResultsFour days after injury, minocycline delivered daily directly into the rabbit knee joints decreased acute chondrocyte apoptosis by 56% compared to controls. Analysis performed six weeks after injury demonstrated superior chondrocyte cell number, cartilage thickness, and cartilage repair in animals receiving short-term (one-week) minocycline treatment compared to controls.ConclusionsThese data support a therapeutic approach utilizing drugs like minocycline for the acute treatment of osteochondral injuries.     

Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles

The natural process of endochondral bone formation in the growing skeletal system is increasingly inspiring the field of bone tissue engineering. However, in order to create relevant-size bone grafts, a cell carrier is required that ensures a high diffusion rate and facilitates matrix formation, balanced by its degradation. Therefore, we set out to engineer endochondral bone in gelatin methacrylamide (GelMA) hydrogels with embedded multipotent stromal cells (MSCs) and cartilage-derived matrix (CDM) particles. CDM particles were found to stimulate the formation of a cartilage template by MSCs in the GelMA hydrogel in vitro. In a subcutaneous rat model, this template was subsequently remodeled into mineralized bone tissue, including bone-marrow cavities. The GelMA was almost fully degraded during this process. There was no significant difference in the degree of calcification in GelMA with or without CDM particles: 42.5 ± 2.5% vs. 39.5 ± 8.3% (mean ± standard deviation), respectively. Interestingly, in an osteochondral setting, the presence of chondrocytes in one half of the constructs fully impeded bone formation in the other half by MSCs. This work offers a new avenue for the engineering of relevant-size bone grafts, by the formation of endochondral bone within a degradable hydrogel.     

An arthroscopic device to assess articular cartilage defects and treatment with a hydrogel

The hydraulic resistance R across osteochondral tissue, especially articular cartilage, decreases with degeneration and erosion. Clinically useful measures to quantify and diagnose the extent of cartilage degeneration and efficacy of repair strategies, especially with regard to pressure maintenance, are still developing. The hypothesis of this study was that hydraulic resistance provides a quantitative measure of osteochondral tissue that could be used to evaluate the state of cartilage damage and repair. The aims were to (1) develop a device to measure R in an arthroscopic setting, (2) determine whether the device could detect differences in R for cartilage, an osteochondral defect, and cartilage treated using a hydrogel ex vivo, and (3) determine how quickly such differences could be discerned. The apparent hydraulic resistance of defect samples was ~35% less than intact cartilage controls, while the resistance of hydrogel-filled groups was not statistically different than controls, suggesting some restoration of fluid pressurization in the defect region by the hydrogel. Differences in hydraulic resistance between control and defect groups were apparent after 4 s. The results indicate that the measurement of R is feasible for rapid and quantitative functional assessment of the extent of osteochondral defects and repair. The arthroscopic compatibility of the device demonstrates the potential for this measurement to be made in a clinical setting.