Purpose

This study compared the macroscopic, histological, and scanning electron microscopy (SEM) characteristics of Chondro-Gide^®, MaioRegen^®, and poly-D,L-lactide-co-caprolactone (PLCL) cell-free scaffolds enhanced with small-diameter microfractures (SDMs) for osteochondral defects (OCDs) in a rabbit model.

Methods and Materials

In total, 54 knees from 27 rabbits were used in this study. Three rabbits were sacrificed at the beginning of the study to form an intact cartilage control group (group IC). An OCD model was created at the center of the trochlea and SDMs were generated in 24 rabbits. Rabbits with OCDs were divided into four groups (n=6 knees per group) according to the cell-free scaffold applied: Chondro-Gide^® (group CG), MaioRegen^® (group MA), PLCL (group PLCL), and a control group (group SDM). Half of the rabbits were sacrificed at 1 month after treatment, while the other half were sacrificed at 3 months after treatment. Healed cartilage was evaluated macroscopically (using International Cartilage Repair Society [ICRS] classification criteria) and histopathologically (using modified O’Driscoll scores and collagen staining). Additionally, cell-free scaffold morphologies were compared using SEM analysis.

Results

The histological characteristics of group IC samples were superior to those of all other groups, except group PLCL, at 3 months after treatment (p<0.05). In addition, the histological properties of group PLCL samples were superior to those of group SDM samples at both 1 and 3 months after treatment. Concerning type 2 collagen staining intensity, the groups were ranked from highest to lowest at 3 months after treatment, as follows: group PLCL (30.3±2.6)>group MA(26.6±1.2)>group CG(23.3±2.3)>group SDM(18.9±0.9). SEM analysis showed that the PLCL scaffold elasticity facilitated transmission of mechanical signals to adherent cells; scaffold pores also aided cell attachment and proliferation.

Conclusion

OCDs treated with enhanced SDM using cell-free PLCL scaffolds had superior histopathological and microenvironmental properties, more hyaline cartilage, and more type 2 collagen, compared with those treated using Chondro-Gide^® or MaioRegen^® scaffolds.

Purpose

In skeletally immature individuals, the covering of bone ends includes a layer of epiphyseal and articular cartilage known as the articular–epiphyseal cartilage complex (AECC). Although initially a contiguous cartilaginous element, the AECC encompasses tissue that becomes the articular cartilage, growth plate, and a secondary ossification center (SOC). During postnatal development joint size increases and cartilage thickness decreases, however the mechanisms that dictate cartilage maturation and joint shaping remain undetermined. This study aims to (1) illustrate and qualitatively compare shape changes of the bone-cartilage interface and (2) quantitatively assess and model volumetric growth of the distal femoral SOC during normal murine postnatal development.

Methods and Materials

With IACUC approval, male wild-type CD1 mice (P0, P7, P14, P21, P28, P35, and P42; n=2-3/age) were sacrificed, perfusion fixed, skinned and eviscerated, then dehydrated to 70% ethanol before imaging by micro-computed tomography at (9 μm)³ voxel resolution. Volume of the left distal femur SOC was calculated by semi-automatic segmentation. SOC volume (V(t)) was fit to a Gompertz sigmoid model (V(t)=V(t=P₄₂)*exp(-exp(-k(t-T))) with 3 parameters (V(t=P₄₂): asymptote/mature value, T: time at inflection, k: growth rate coefficient).

Results

Although the SOC was not evident at P0, it increased in size between P7 (0.3±0.1 mm³ ) and P42 (~1900%; 6.4±0.1 mm³ ). The SOC initially formed as a small ellipsoidal structure followed by protrusion of the condyles, and formation of the trochlear ridge and intercondylar notch. Increase in SOC volume followed Gompertz function (R²=0.98, RMSE = 0.4), such that growth was slowest at the start and end of the time assessed, with an inflection point at T=~11 days postnatal and growth rate coefficient of k=0.23 1/days.

Conclusion

Growth of the murine distal femoral SOC during postnatal development follows a sigmoid model indicating 3 phases of growth: the initial lag, the log, and the diminishing growth phases, where mechanisms of growth and maturation may vary

Purpose

Single-cell sequencing has potential to provide unprecedented insight into cell-to-cell communication and complex cross-talk between joint tissues. However, difficulty remains in preparing entire joints for comprehensive scRNA-seq analysis due to the great diversity of tissues. Joints tissues like bone and cartilage consist of calcified weight-bearing matrix sparsely populated by cells, whereas synovium and fat pads are highly cellular tissues, and immune cells may have little or no surrounding matrix. To capture this broad range of cell types we developed a two-stage digestion protocol.

Methods and Materials

Mouse knees from femoral and tibial growth plates were isolated, minced and digested in two stages. To protect softer cells from over-digestion a gentler 1% collagenase-IV (30 mins) digestion was used first. An aggressive digest, 2% collagenase-II (90 mins) followed to free cells within cartilaginous and bony matrix.

After each digest, released cells were strained, and RBCs lysed. 10X Chromium Single Cell Gene Expression platform and Illumina HiSeq 4000 were used. Cell Ranger and Seurat created PCA clustering.

Results

This sequential protocol demonstrated consistency of gene expression between contralateral knees by similar cell-types (Fig. 1a). Both digests are required for comprehensive cell-type identification and inclusion (Fig. 1b).

A diverse collection of cells was captured by this protocol, with many cell-types from hard and soft tissues shown here by selected signature genes (Fig. 2).

Conclusion

To understand the joint environment, we must understand the roles of all cells present in-vivo. This complex problem is currently best studied using scRNA-seq to assess what is happening at the cellular level. This protocol will be valuable for understanding the in-vivo conditions in which cartilage resides and provide unique insight for the study of joint preservation with age or injury.

Purpose

Chronic, low-grade inflammation (inflammaging) contributes to the age-dependent deterioration of chondrocytes, or chondrosenescence. The chondrosenescent phenotype includes the development of a senescence associated secretory phenotype (SASP). Using both simulated aging (monolayer expansion) and inflammation, we sought to create an in vitro model that replicates the chondrosenescent phenotype found in patient samples. We hypothesized that treatment of young human articular chondrocytes (HACs) with inflammatory cytokines during monolayer expansion will induce biomarker profiles and ECM production levels similar to that of HACs isolated from aged/OA cartilage.

Methods and Materials

HACs (30 ± 10 years) were treated with 1 ng/mL IL-1β during monolayer expansion. Expression of senescence markers (SA-β-gal and p16) and RNAseq for SASP were measured at each passage. High passage, chondrosenescent HACs were then redifferentiated in pellet culture for 28 days and sGAG production was measured using the DMMB assay and Alcian blue staining.

Results

IL-1β treatment during expansion reduced proliferation (Figure 1), increased expression of SASP factors (Figure 2), and increased expression of senescence markers SA-β-gal and p16 (Figure 1) after 7 passages. SASP markers including MMPs, ILs and CXCLs were higher in IL-1β treated samples at P8 (Figure 2a). Expression of SASP markers decreased over time but remined 2-3 orders of magnitude higher in IL-1β treated samples. (Figure 2b). When redifferentiated in pellet culture, matrix production was lower in treated and untreated P7 chondrocytes compared to P4. IL-1β treatment reduced ECM production to levels comparable to OA chondrocytes (not shown).

Conclusion

Treatment with IL-1β during monolayer expansion decreased proliferative potential of HACs after the 4^th passage and accelerated expression of SASP and other senescence markers, making it a viable system to model and study chondrosenescence.

Purpose

Osteoarthritis (OA) is the most common degenerative joint disease without clear pathophysiological mechanism and effective drugs for treatment. Though various animal models are existing, the translation of these models into clinics remains difficult due to species differences. To advance the clinical relevance of research data, a preclinical OA model with human samples is indispensable.

Methods and Materials

In this study, an ex vivo inflammatory OA model was induced using explants from human OA femoral head. Effects of treatment with different concentrations of IL-1β and TNF-α were compared after 3- and 7-day culture duration. The samples were assessed histologically (n = 3/group), biochemically (n = 6-11/group), and transcriptionally (n = 6-8/group).

Results

In the inflammatory OA groups, the gene expression levels of cartilage catabolism (MMP1, MMP3), and inflammation (IL-6, IL-8) markers were significantly upregulated, while the anabolic genes (COL2, ACAN, PRG4) were downregulated compared with the control group. The release of cytokines (IL-6, IL-8) and nitric oxide (NO) in conditioned medium was also upregulated in the inflammatory OA groups. The Safranin O/Fast Green staining showed that the degradation of proteoglycan in the superficial zone cartilage presented a positive correlation with culture duration and concentration of IL-1β and TNF-α. Cell viability in the cartilage tissue was > 85% and comparable among all groups. For establishment of human osteochondral explant inflammatory OA model, 1 ng/mL IL-1β + TNF-α were enough to induce a mild inflammatory OA condition; 5 ng/mL IL-1β + TNF-α induced a more profound inflammatory and catabolic effect; 10 ng/mL IL-1β + TNF-α showed a similar effect as 5 ng/mL.

Conclusion

The results indicate that an ex vivo OA model of inflammation and degeneration was successfully established using osteochondral explants from human femoral head. This model can be used to elucidate the in-depth mechanism of inflammatory OA, and to screen new drugs for its treatment.

Purpose

In vitro modelling of human cartilage is commonly used to understand injury and disease in the tissue and to test treatment options. Organ-on-a-chip (OOAC) is an emerging modelling technology, incorporating cell culture chambers and microfluidics, that seeks to improve current modelling capabilities by increasing physiological relevance. Here, we assess the Pregenerate OOAC system to investigate human chondrocyte responses to pro-inflammatory stimuli cf. traditional chondrocyte culture techniques.

Methods and Materials

The Pregenerate OOAC consists of a central cell chamber (volume: 7.5µl) connected to a medium channel with an inlet at either end; six of such culture systems are contained on one chip (Figure 1A). Chondrocytes were isolated from macroscopically intact cartilage obtained during knee arthroplasty (n=5) and culture-expanded in monolayer to passage 2. Chondrocytes were encapsulated in Fibrin hydrogel and 2,200 chondrocytes loaded into each cell chamber of the microfluidic chips. Parallel cultures were set in high-density pellets (200k cells/pellet) or in monolayer (5000 cells/cm²). After 21 days, cultures were stimulated with a pro-inflammatory cocktail (50ng/ml TNF-α, 10ng/ml IL-1β and 25ng/ml IFN-γ) for 24 hours prior to assessing gene expression levels of COL1A1, COL2A1, COL10, ACAN, SOX9, MMP1, MMP3, MMP13 and ADAMTS-5.

Results

Fluorescence microscopy demonstrated evenly distributed chondrocytes within the culture chamber (Figure 1B). Pro-inflammatory stimulation down-regulated COL1A1, COL2A1, ACAN and SOX-9 in monolayer and pellet cultures only; COL10 was down-regulated in monolayer and pellet cultures but up-regulated in the Pregenerate chip. MMP3 and MMP13 were up-regulated in all three culture systems. ADAMTS-5 was down-regulated in the chips only. There was no change in expression of MMP1 in any of the culture systems.

Conclusion

These results demonstrate distinct differences in human chondrocyte responses to pro-inflammatory stimuli in the Pregenerate microfluidic chip cf. traditional culture conditions. Further work is needed to investigate these differences and which system is more synonymous with in vivo behaviour.

Purpose

There is an unmet need for novel, disease-modifying pharmaceuticals for the treatment of osteoarthritis (OA). The development of such therapeutics is hampered by a lack of physiologically relevant preclinical models, which contributes to significant attrition in the drug development pipeline. Organ-on-a-chip technology can be utilised to generate new in vitro models with a greater capacity to replicate human physiology in health and disease. Our aim is to develop an organ-on-a-chip model of the human synovial joint.

Methods and Materials

A commercially available, two-channel, microfluidic chip (Chip-S1®, Emulate Inc) was used to develop the joint-on-a-chip model (Figure 1A). Primary human fibroblast-like synoviocytes (FLS) were cultured in the top channel within 10% Matrigel to replicate the intimal synovial lining layer, and primary human chondrocytes were cultured in the bottom channel within 2% agarose to mimic the articular cartilage (Figure 1A). After 24h, cell viability in the chip was assessed by live/dead (calcein AM/ethidium homodimer 1) staining. After 72 hours, cell morphology and cytoskeletal architecture was assessed by actin labelling.

Results

FLS and chondrocyte viability was maintained at 24h (Figure 1B) and exhibited appropriate cytoskeletal architecture at 72h (Figure 2). Chondrocytes demonstrated a characteristic spherical morphology and were distributed evenly throughout the agarose. FLS were fibroblastic and formed a single layer of cells within the top channel of the chip, over the dividing membrane (Figure 2).

Conclusion

The work presented here represents the early stages of development of a human synovial joint-on-a-chip and suggests that this model, built upon the Emulate Chip-S1®, is viable. Further validation of the model is currently ongoing. The Emulate system allows for the application of mechanical stimulation in the form of fluid shear and tensile strain and has numerous potential applications. For example, synovial inflammation can be mimicked through the addition of pro-inflammatory cytokines and drug (e.g. steroid) therapeutic efficacy can be screened.

Purpose

In vitro cell culture and tissue engineering techniques often rely on animal sera such as foetal bovine serum (FBS) or on human serum (HS). However, reproducibility of experiments and applicability of the results on in vivo situations is limited. Furthermore, there are ethical concerns (1) with the production of FBS via killing calves in the womb, and (2) the use of blood donations for the production of HS for research. Hence, the aim of this study was to compare the differentiation degree of cartilage-like microtissues cultivated with species specific sera and a chemically defined serum (CDS).

Methods and Materials

Human primary chondrocytes isolated from knee joints were expanded in medium containing HS. Scaffold-free microtissues were generated and cultivated in cell-repellent plates using three different sera: HS, FBS and CDS. After two and four weeks, the macroscopic appearance (reflected-light microscopy) was documented and the size was determined. The differentiation degree was evaluated via histology to visualize typical glycosaminoglycans (Safranin O, Alcian blue) and immunohistochemistry (IH) to detect cartilage-specific markers (collagen type II, proteoglycans, Sox9) and collagen type I on cryosections.

Results

All microtissues had a whitish colour both after two and four weeks cultivation. Cartilage-like microtissues cultivated with HS or FBS were similar in size, whereas those cultivated with CDS were more than 10% bigger. Already after two weeks, cartilage-like microtissues of all tested sera showed an expression of cartilage-specific markers, which increased after four weeks. Collagen type I was always weakly expressed. The expression level of all tested markers was similar regardless of serum type.

Conclusion

This study suggests that the differentiation degree of cartilage-like microtissues in cell-repellent plates is not influenced by the serum type. Hence, CDS can replace species specific sera to reduce ethical concerns and batch-to-batch variations. The applicability of CDS to the expansion of chondrocytes remains to be determined.

Purpose

Traumatic joint injury can initiate osteoarthritis and launch a slow process of creeping mineralization and tidemark advancement through unclear mechanisms. High-frequency ultrasound is a non-destructive approach that can be used to image the cartilage-bone interface. The purpose of this study was to develop a novel quantitative ultrasound (QUS) parameter that reflects alterations in tidemark structure due to changes in mineralization.

Methods and Materials

Osteochondral explants 3mm in diameter were extracted with 8G Jamshidi needles from an adult bovine shoulder (N=1) or intact human knee cartilage (N=3, de-identified arthroplasty surgical waste). Explants were cultured in vitro, fixed and decalcified in EDTA. Specimens were SAM scanned by raster-scanning a 15 MHz transducer (Olympus) at the optimal focal length of 50.8 in a 6x6mm grid at 100 µm steps. A log-compressed envelope signal (dB per mm) in a 1 mm² area in the center of each explant was produced (MATLAB). The QUS alpha parameter was defined as the slope (dB/mm) between the maximal subchondral signal and 200 µm prior. Differences were evaluated by matched pair design (JMP-Pro, significance set at p˂0.05).

Results

Osteochondral SAM scans produced two prominent signals along the scanning axis at the articular cartilage surface and the tidemark (Fig. 1A), as previously reported by others. We report the novel finding that decalcification altered the echogenic backscatter at the tidemark, with increased echogenicity in subchondral bone (Fig. 1B). Compared to healthy bovine tissue, human OA articular cartilage was thicker, produced more backscatter inside the articular cartilage, showed higher alpha values, and required longer decalcification times to observe changes in alpha.

Conclusion

A high alpha indicates a rapid spatial increase in echogenicity that is expected for a well-developed and intact mature tidemark. Tidemark advancement produces a hypomineralized tissue suggesting that QUS could be useful for identifying longitudinal changes in the tidemark, in an explant model.

Purpose

The Missouri Osteochondral Allograft Preservation System (MOPS) has been shown to extend the period of acceptable cell viability compared to the standard of care (SOC). Metabolic changes and mechanisms by which cells remain viable cannot be evaluated using common viability assessments. This study aimed to compare metabolic changes in chondrocytes after storage in MOPS or SOC conditions using a tri-stain method.

Methods and Materials

Ovine femoral condyles were stored using MOPS (proprietary media, 22-25°C storage) or SOC [Lactated Ringer’s solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage]. Cartilage was stained concurrently with Calcein-AM, Ethidium Homodimer-1, and Mitotracker and imaged using a confocal microscope following dissection (fresh control) or after 56 days of storage. Morphological features, including presence of a Mitotracker boundary (Fig. 1B-E) was used to develop a grading system (Fig. 1) ranging from Grade-1, representing highly metabolic and viable cells, to Grade 5, representing dead cells with no metabolic activity. Results were reported as percentage of total cells per grade, with % live calculated by summation of Grades 1 and 2 (no Ethidium Homodimer present). T-tests were performed.

Results

1513 cells were graded in 9 samples (Table 1). When compared to SOC, MOPS samples displayed higher percentage of living cells and Grade-2 cells, as well as less Grade-3 and Grade-4 cells. When compared to controls, both 56-day storage groups had significantly lower Grade-1 cells and significantly higher Grade-5 cells. The mechanism causing the Mitotracker boundary (Fig. 1B-E) is unknown with a possible explanation of intracellular organelle transfer due to cell stress signaling.

Conclusion

MOPS maintained more viable chondrocytes than SOC after 56 days of storage. The grading system revealed changes in metabolic activity and viability resulting from storage. The three-stain approach and novel grading system allowed categorization beyond the live/dead binary and may contribute to understanding how chondrocytes respond to prolonged storage.

Purpose

In the early stages of cartilage damage, development of diagnostic methods focusing on the mechanism of maintaining hydrostatic pressure of cartilage is thoughted to be useful. On the other hand, 17O-labeled water, which is a stable isotope of oxygen, has the advantage of being free from radiation exposure and allergic reactions, and can be detected as the dynamics of water molecules by MRI. The purpose of this study is to evaluate MRI images using 17O-labeld water in a rabbit model.

Methods and Materials

Anterior cruciate ligament transection surgery was performed on 14-weeks-old male Japanese white rabbit. The lower legs were harvested at 4- and 8-weeks postoperatively, and MRI imaging, macroscopic and histological evaluation were performed. Using 3T MRI scanner, T2-weighted images using the 2D-fast spin echo method were performed repeatedly 18 times. 3 scans after the dynamic scanning, 17O-labeled water was manually administered via catheter. Multiple small regions of interest (ROI) were set in the articular cartilage of both condyles referring to the macroscopic or histologically apperance. Concentration of 17O in each phase of each ROI was calculated using the signal value of the T2-weighted image. The time course of concentration of 17O in each ROI was evaluated between the macroscopic and histological grades. All results were statistically compared using one-way ANOVA and significance was accepted with a p value < 0.05.

Results

An increase in novel contrast medium concentration was observed by MRI, consistent with macroscopic and histologically injured areas.
The macroscopic evaluation between the two groups showed that the concentration of 17O increased significantly after Phase 10 compared to the control area. Histologic evaluations also showed that the concentrations of 17O significantly increased in the group of OARSI grading 3 compared with that of other grades.

Conclusion

17O-labeled water could visualize earlier articular cartilage damage, which is difficult to detect by conventional methods.

Purpose

There is a clinical demand for a tool for peri-surgical, non-destructive evaluation of local cartilage quality. Such a tool can be used for evaluation of treatment necessity, determination of (viable) borders of a cartilage defect, and evaluation of repair tissue during second-look surgeries. Therefore, the aim of this study was to assess the feasibility of near-infrared spectroscopy as a tool for non-destructive, minimally-invasive measurement of cartilage quality.

Methods and Materials

A custom-developed 2-mm optical-fibre probe was attached to a 350-1830nm wavelength spectrometer. 450 diffuse reflectance spectra were obtained from 90 cartilage sites on eight fresh-frozen human cadaveric knees. Local glycosaminoglycan, hydroxyproline and water content; cartilage thickness; Young’s modulus (using micro-indentation); T1rho and T2-mapping MRI images; and macroscopic osteoarthritis score were obtained. With 35 spectral features, related to water, lipids and blood, a linear regression model was used to assess the relation to physiological outcome parameters. Moreover, a linear discriminant classification model was used to study the discriminative power between weight-bearing and non-weight-bearing areas. Additionally, in a mini-pig cartilage-defect model, the difference between defect and nearby control areas (40 measurements over 8 defects) was classified directly post-mortem.

Results

The linear models showed the best correlation with osteoarthritis score (R²=0.72), cartilage thickness (R²=0.70), and hydroxyproline content (R²=0.61), and the weakest with Young’s modulus (R²=0.42). Higher correlation to T2 mapping than T1rho (R²=0.55 vs 0.45) was found, suggesting higher sensitivity to matrix organization than concentration. Weight-bearing and non-weight-bearing parts were classified with high accuracy (90.9%). Spectral measurements in- and outside the cartilage defects in the mini-pigs (Fig. 1) could be classified with 100% accuracy.

Conclusion

Good correlations between spectral features and osteoarthritis-related measures were found. Further validation in vivo is needed. The discriminative classification between healthy and damaged cartilage was excellent, suggesting that applications for cartilage defect (repair) evaluation are feasible.