Purpose

Autologous chondrocyte implantation (ACI) is a worldwide recognized therapy to treat focal cartilage lesions. Here we describe the experiments followed by our group to develop a modification of MACI called High Density-Autologous Chondrocyte Implantation (HD-ACI) in which cell density is increased 5-fold.

Methods and Materials

In classical ACI, 5 million of cultured chondrocytes were implanted in the cartilage defect, but the use of periosteum increased morbidity. In MACI, 20 million cells are seeded in a 20 cm2 collagen membrane, so cartilage defects are treated with a density of 1 million cells per cm2. Surgeons observed that neo-formed tissue was softer than normal cartilage and had scarce cell number. We proposed to increase cell density to 5 million cells per cm2, and first of all we had to prove its safety and effectiveness in a sheep model. In these experiments, 1 million and 5 million chondrocytes as well as 5 million mesenchymal stem cells were tested in 1 cm2 defects performed in the cartilage of the medial femoral condyle. In all animals, we carried-out other cartilage defects that were repaired with microfractures.

Results

Histological and molecular studies demonstrated that the animals treated with 5 million chondrocytes regenerated cartilage defects with a better hyaline cartilage than those treated with 1 million cells while fibrocartilage was produced in the animals treated with mesenchymal stem cells and in the defects treated with microfractures. Currently, 336 patients (251 in the knee, 82 in the ankle and 3 in the hip) have been treated with HD-ACI and in this work, we present the results of 176 treated in the knee and 48 in the ankle

Conclusion

Our results demonstrate that HD-ACI is a safe and effective technique for the treatment of cartilage defects, giving rise a neo-formed hyaline tissue and improving clinical and subjective perception of the joint functionality

Purpose

Fresh allografts are typically used in chondral allograft transplantations but are commercially available for only 28 days. Cryopreservation is a promising alternative that can effectively extend storage time, thereby increasing donor tissue availability. This study will investigate the use of fresh and vitrified particulate articular cartilage allograft transplantations in porcine knee joints by comparing mechanical properties of the cartilage after transplantation.

Methods and Materials

Transplantations of particulate articular cartilage into 10mm diameter cartilage defects were performed on n=19 porcine femoral condyles of sexually mature female pigs. Experimental groups included control, fresh, vitrified, and negative control. The fresh group included fresh particulate cartilage (n=8 replicates) stored at 4°C for 3 weeks. Vitrified particulate cartilage (n=8 replicates) was subjected to a 2-step 30-minute vitrification protocol and stored in liquid nitrogen at -196°C. Before transplantation, the vitrified cartilage was warmed in a 37°C water bath for ~30 seconds and washed in DMEM-F12 to remove cryoprotectant. In the negative control group, fibrin glue without particulate cartilage (n=3 replicates) was used. After transplantation, the pigs were given ~6 months (±3 weeks) for their condyles to heal, then the 10mm diameter osteochondral dowels were harvested along with healthy control dowels from the contralateral knee joints. Unconfined compression testing was performed on all samples in 3 cycles of rapid loading (0-10%, 10-20%, 20-30% strain) and holding the displacement for 15 minutes to allow for stress-relaxation after each cycle.

Results

The instantaneous modulus, equilibrium modulus and relaxation time constants (Prony series) of the vitrified samples were similar to the fresh samples and statistically significantly stiffer (equilibrium modulus p-value<0.05) than the negative controls. The vitrified cartilage had slightly higher values than the fresh, making it closer to the control group without cartilage defects.

Conclusion

This study provides mechanical evidence that vitrified particulate cartilage allografts are a promising cartilage source for articular cartilage defect repair.

Purpose

To support the preclinical development of novel treatments for cartilage injury, we generated a transgenic rat strain that can noninvasively report chondrogenesis for correlation with endpoint measures of tissue repair.

Methods and Materials

All animal studies were pre-approved by the local institutional ethics committee. A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. A transgenic line was established on a Lewis background and characterized by serial bioluminescence imaging and ex vivo measurement of reporter levels. The sensitivity and specificity of the strain were assessed using an osteochondral defect model in 6 month old or 2 year old rats. To stimulate healing, some defects were treated with rat bone marrow stromal cell aggregates.

Results

Bioluminescence imaging of the strain revealed substantive signal from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance declined with postnatal development yet remained detectable in aged animals. Explant imaging, immunohistochemistry, and gene expression confirmed that both firefly luciferase and mCherry expression were localized to cartilage. Implantation of wild type bone marrow stromal cells into osteochondral defects made in young adult reporter rats led to a temporal elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair (Figure 1). For aged rats, higher variability in bioluminescence signal was associated with generally poor repair of the cartilage phase with some restoration of the osseous phase. While mCherry-positive cells were detected in aged repair tissue, senescence associated β-galactosidase and p16^INK4A immunostaining indicated the presence of abundant senescent cells (Figure 2). Ongoing studies are determining the influence of senolytic drug delivery on reporter activity and cartilage formation.

Conclusion

The reporter strain can serve as an initial preclinical model for testing strategies to stimulate chondrogenesis within articular cartilage defects.

Purpose

Synovial joints are essential for body movement. Unfortunately, articular cartilage (AC) is highly susceptible to disease and exhibits poor repair capacity that current clinical strategies fall short of rectifying. To improve these strategies, more information is needed on AC development and, specifically, how a functional multizone organization is acquired. Mature AC consists of: flat, lubricant-producing surface zone cells; round, column-aligned, and load-resistant deep zone cells; and a mineralized zone of cells demarcated by the tidemark. How this organization is generated during postnatal growth remains poorly understood. To explore mechanisms, we asked whether and how primary cilia regulate AC morphogenesis. Primary cilia are mechanical- and morphogen-transducing cell surface organelles that are crucial in morphogenesis of many mammalian tissues. Their roles in growth plate chondrocytes are well appreciated and functions have been implicated in AC but remain unclear.

Methods and Materials

We used a conditional loss-of-function approach (Ift88-flox) targeting joint-lineage progenitor cells (Gdf5Cre) and monitored structural/functional consequences on postnatal knee AC development.

Results

We found that embryonic joint development and growth up to 3 weeks of age were largely unaffected in mutants. However, mature (8 weeks) tissue exhibited: highly disorganized extracellular matrix (ECM) (i.e. aggrecan and collagen2); disrupted tidemark patterning and zonal organization; and markedly reduced mechanical properties (AFM-based testing). Further analyses revealed zonal disorganization was likely driven by disorganized chondrocyte differentiation (not degradation) and that hedgehog signaling was markedly disrupted in mutant joints. Notably, the changes in hedgehog response gene patterns related to, and likely caused, changes in tidemark topography and regional ambulatory load responses, increasing dramatically in loaded regions of the mutant tibial plateau. Interestingly, Prg4 expression was also increased in those loaded sites.

Conclusion

Overall, our data provide clear evidence that primary cilia orchestrate -and are essential for- postnatal AC morphogenesis, dictating tidemark topography, zonal chondrocyte composition and responses to local ambulatory loads.

Purpose

Autologous chondrocytes implantation is a common procedure for large cartilage defect, which takes two-time surgeries and high-cost cell processing centers. Recently, we revealed that the systemic administration of high mobility group box-1 peptide (HMGB1-44) has a potential to accelerate scarless tissue regeneration via circulating mesenchymal cells. The purpose of this study was to investigate the effects of HMGB1-44 peptide to murine osteochondral defect.

Methods and Materials

HMGB1-44 peptides were intravenously administered (100 μg in 100 μL saline) 2 times a week into 8-week-old adult male mice with 0.5 mm depth and 0.5mm width osteochondral defect. After 4 weeks administration, Wakitani score was compared with vehicle (100μl saline) group. Furthermore, to reveal the mechanism, we evaluated induced tomato-fluorescent-positive cells in blood and defect sites of wild type mice parabiotically paired with PDGFRa-Cre; ROSA^tdTomato mice. Also, the immunohistochemistry with Col1, Co2, Sox9, and Acan were evaluated at 12 weeks after osteochondral injury. Finally, to investigate the relationship with Cxcr4/Cxcl12 axis, efficacy of AMD3100, Cxcr4-specific antagonist, was examined.

Results

While saline administration resulted in a failure of hyaline cartilage regeneration and deposition of fibrous scar tissues (Fig.1), HMGB1-44 peptide administration significantly induced the regeneration of the hyaline cartilage stained with safranin-O, and their Wakitani score were higher than those of vehicle group. The accumulation of tomato-fluorescent-positive cells were higher in blood and defect sites in HMGB1-44 group. Also, tomato-fluorescent-positive cells marked higher Col2, Sox9, and Acan and lower col1 expressions (Fig2). Finally, the accumulation of tomato-fluorescent-positive cells reduced by AMD3100 and cartilage regeneration with hyaline cartilage was cancelled by AMD3100.

Conclusion

HMGB1-44 peptide induced circulating mesenchymal cells, which expressed Sox9, Col2 and Acan in osteochondral defect sites, via CXCR4/CXCL12 axis. Intravenous administration of HMGB1-44 peptide induced hyaline cartilage regeneration and repaired murine osteochondral defect

Purpose

The pathogenesis of osteoarthritis often begins from an injury such as a focal defect to articular cartilage, which establishes chronic, low-grade inflammation that eventually results in degenerative joint disease. Pluripotent stem cell-derived articular chondrocytes represent a promising new tool for articular cartilage repair.

Methods and Materials

Research-grade human pluripotent stem cells (hPSC) were differentiated into immature articular chondrocytes using protocols previously established in our lab. Generated hPSC-derived chondrocytes (hPSDC) were seeded onto clinical grade collagen 1/3 membranes (hPSDC-M) and evaluated using single-cell RNA-sequencing to characterize and assess the developmental status. Potential of engraftment of the hPSDC-M was assessed in a porcine model, where 6-mm full thickness defects were generated in the articular cartilage of male Yucatan minipigs.

Results

Our studies have shown that transcriptional signatures of hPSDC-M via single cell sequencing closely resemble embryonic and juvenile stages of human chondrocyte ontogeny, suggesting an immature chondrogenic identity. Our porcine model also demonstrated that implanted hPSDC-M engrafted and contributed to the generation of comparable articular cartilage tissue endogenously found when compared to the membranes alone. Engrafted human chondrocytes were clearly detectable at 6 months after implantation in all tested animals. Biomechanical assessment after 6 months confirmed that the cartilage containing the hPSDC-M showed significant improvement in cartilage surface biomechanical properties compared to the membrane alone. which, and positively stained for GAGs, PRG4, collagen 2, and SOX9 via IHC. Human hPSDC-M secreted nearly 10-fold more BMP2 than BMSCs indicating that pluripotent stem cells closely resemble chondroinductive signature of early perichondrium, while adult MSCs lacked this potential.

Conclusion

Both the functionality by engraftment of these immature chondrocytes and their paracrine activity contribute to the value of hPSDC-M, which has the potential to enact articular cartilage repair as a universal, off-the-shelf product from an inexhaustible source of chondrogenic cells.

Purpose

The zonal properties of articular cartilage contribute to the biphasic mechanical properties of the tissue. Current cell-based therapy with autologous chondrocyte implantation (ACI) has not been able to regenerate the zonal architecture with resulting tissue degeneration in the long-term. Stratified implantation of zonal chondrocytes has been hypothesized to improve the recapitulation of zonal architecture and long-term integrity of regenerated tissues. In this study, the efficacy of bilayered implantation of autologous zonal chondrocytes in achieving cartilage regeneration was investigated in a critical size porcine chondral defect model.

Methods and Materials

Autologous zonal chondrocytes were derived by integrating dynamic microcarrier (dMC) expansion followed by high-throughput microchannel size-based cell separation (Figure 1). Expanded zonal chondrocytes were implanted as bilayered fibrin hydrogel of superficial zone (SZ) chondrocytes overlaying middle/deep zone (MZ/DZ) chondrocytes in femoral condyle defects, with a single layer of full-thickness chondrocytes as control. Harvested tissues were subjected to histology, polarised light microscopy, MRI, microCT and mechanical analysis.

Results

The zonal chondrocyte production strategy was able to generate sufficient numbers of high-quality zonal chondrocytes from 0.5 cm² of clinically relevant non-weight-bearing cartilage plugs for treatment of ≥ 5 cm² of cartilage lesion, overcoming the challenges of limited autologous chondrocyte availability in the clinical setting and loss of zonal cartilage phenotype during expansion. Six months post-implantation, compared to implant with either tissue culture plate or dMC-expanded full-thickness chondrocytes, the bilayered implantation of dMC-expanded zonal chondrocytes improved cartilage regeneration with a substantial recapitulation of zonal architectures, including chondrocyte arrangement, specific PRG4 distribution and collagen alignment. The recapitulation of stratified architecture in regenerated tissues was accompanied by healthier underlying subchondral bone structure.

Conclusion

With the appropriate zonal chondrocyte isolation and expansion strategy, the approach of stratified zonal chondrocyte implantation represents a significant advancement over the current ACI-based cartilage regeneration, with the potential to improve the long-term integrity of regenerated cartilage tissues.

Purpose

This study investigated the effect of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) in an ovine model of early osteoarthritis (OA).

Methods and Materials

Fourteen female, 3-4 year old Welsh Mountain sheep underwent medial meniscal transection in their left stifle joints. Four weeks post-surgery, 10⁷ Quantum® bioreactor-culture expanded hUC-MSCs (pooled from 3 donors) in 50µL PBS (n=7) or PBS alone (n=7, vehicle control group) were injected into the knees. Joints were harvested 12-weeks post-surgery and underwent X-ray and Magnetic Resonance Imaging (MRI) and histological processing. Scoring was performed via a macroscopic OA score, Kellgren-Lawrence score (X-ray), sMOAKS (whole joint on MRI) and pyKNEEr (cartilage thickness on MRI), and cartilage and synovitis histology scores (Table 1).

Results

There was an improved macroscopic OA joint score in sheep receiving hUC-MSCs (11±4) cf. control (14±6), but this did not quite reach significance (p=0.054; Table 1). Kellgren-Lawrence X-ray scores in joints treated with hUC-MSCs were significantly improved (2.0±0) cf. control (3.0±0; p=0.028). No significant difference in sMOAKS (hUC-MSCs 18±10 vs. no cells 22±9; p=0.784) or PyKneer (hUC-MSCs 0.8±0.42mm vs. no cells 0.67±0.33mm; p=0.628) was observed between the treatment groups. Histological scoring demonstrated improved cartilage in sheep receiving hUC-MSCs (37±6) cf. control (41±13), but again, not quite significantly (p=0.064). A low level of synovitis was seen in both treatment groups (hUC-MSCs: 3.0±0.5 vs. no cells 3.0±2.5; p=0.900), i.e. typical of post-surgery, but with no increase caused by injecting xenogeneic cells.

Conclusion

There was significantly less OA observed on X-rays in sheep treated with hUC-MSCs than vehicle control, with no indication of an inflammatory response to the cells at the time-point studied. Whilst corroborating suggestions from previous murine models of OA and injury, that hUC-MSCs have benefit as an allogeneic treatment for early OA, this study would perhaps have benefitted from an increase in numbers or timescale.

Purpose

The lack of cues to guide the chondrogenic differentiation of egressed mesenchymal stromal cells (MSCs) can be attributed to the poor outcomes upon microfracture. This study evaluates the ability of cLIUS when applied at the tissue resonant frequency of 3.8 MHz to yield superior repair outcomes upon microfracture in a rabbit model. The ability of cLIUS to maintain chondroinductive and chondroprotective ability in a pro-inflammatory environment was studied.

Methods and Materials

Bilateral microfracture defects were created on the femoral medial condyle of female New Zealand white rabbits (n=12) and the left joint received cLIUS treatment (3.8 MHz, 3.8 Vpp, 8 minutes/application/day) and the contralateral right joint served as the control (Fig.1A). Rabbits were euthanized at 8-weeks and outcomes were assessed histologically. The ability of cLIUS to maintain MSC chondrogenesis in a proinflammatory milieu was evaluated by assaying for NF-kB pathway markers.

Results

Defect areas in the right joints exhibited irregular cartilage surface and loss of glycosaminoglycan (GAG). The defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte-morphology, and columnar organization (Fig.1B/D). Significantly higher O’Driscoll scores were obtained for the left knee joints (Fig. 1E). No adverse impact on bone or change in the joint space was noted (Fig. 1C). Synovial fluid collected from cLIUS treated left knee joints demonstrated lower levels of cytokines when compared to the synovial fluid untreated right knee joints (Fig. 1F). The presence of TNFα reduced the levels of SOX9 in the nucleus when compared to control and cLIUS samples (Fig. 2). cLIUS stimulation in the presence of TNFα inverted the localization of these markers, where the levels of nuclear pNFκB were decreased and SOX9 was upregulated.

Conclusion

Our results demonstrate that healing of chondral defects treated with microfracture can be accelerated by employing cLIUS regimen that possess chondroinductive and chondroprotective properties.

Purpose

There is an urgent need for a synthetic cartilage implant that can immediately withstand physiologic loading, prevent damage to articulating cartilage, and restore joint function for a wide patient demographic. In this study, a new synthetic bearing material (HYALEX® Cartilage) with bone fixation using an in situ photopolymerized adhesive was assessed in a large animal model.

Methods and Materials

The synthetic bearing is engineered from materials with the same unique structure-function relationships as native hyaline cartilage (Figure 1). An interpenetrating polymer network (IPN) of polyether urethane (PEU) and negatively charged polyelectrolyte is formed during manufacturing. PEU provides strength, while the polyelectrolyte causes the network to become hydrophilic and lubricious under load [Unterman, MRS 2020]. The hydrophobic backside of the synthetic bearing is optimized for fixation using a photopolymerizable adhesive [Bichara, ORS 2020].

Synthetic cartilage devices (8 mm diameter x 2 mm thickness) were implanted in medial femoral condyles of four adult goats. Using a medial parapatellar approach, a 4.1 mm deep osteochondral defect was drilled. Adhesive was applied in the site, the implant was seated, and the adhesive was photopolymerized. Joint tissues and implants were evaluated at 8 weeks.

Results

All implanted animals were fully weightbearing within 24 hours post-operatively, returned to normal gait, and successfully reached the study endpoint. Implants remained intact and well-fixed with no evidence of loosening or subsidence (Figure 2). Peri-implant cartilage and the anterior menisci articulating against the synthetic cartilage IPN were both normal. Medial tibial cartilage was equivalent for operated and contralateral control joints, indicating that articulation with the synthetic cartilage IPN maintained the native condition of the tissue.

Conclusion

A synthetic cartilage implant consisting of a lubricious, cartilage-friendly, hydrated IPN bearing material fixed in situ with a photopolymerizable adhesive has the potential to provide an off-the-shelf option for cartilage lesion repair and joint preservation.