A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Assessment of properties, applications and limitations of scaffolds based on cellulose and its derivatives for cartilage tissue engineering: A review.

Cartilage is a connective tissue, which is made up of ~80% of water. It is alymphatic, aneural and avascular with only one type of cells present, chondrocytes. They constitute about 1-5% of the entire cartilage tissue. It has a very limited capacity for spontaneous repair. Articular cartilage defects are quite common due to trauma, injury or aging and these defects eventually lead to osteoarthritis, affecting the daily activities. Tissue engineering (TE) is a promising strategy for the regeneration of articular cartilage when compared to the existing invasive treatment strategies. Cellulose is the most abundant natural polymer and has desirable properties for the development of a scaffold, which can be used for the regeneration of cartilage. This review discusses about (i) the basic science behind cartilage TE and the study of cellulose properties that can be exploited for the construction of the engineered scaffold with desired properties for cartilage tissue regeneration, (ii) about the requirement of scaffolds properties, fabrication mechanisms and assessment of cellulose based scaffolds, (iii) details about the modification of cellulose surface by employing various chemical approaches for the production of cellulose derivatives with enhanced characteristics and (iv) limitations and future research prospects of cartilage TE.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Complete Assessment of Multilineage Differentiation Potential of Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.

Adherent agarose mold cultures: An in vitro platform for multi-factorial assessment of passaged chondrocyte redifferentiation.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFß1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

Assessment of the effect of systemic delivery of sclerostin antibodies on Wnt signaling in distraction osteogenesis.

Sclerostin is a known inhibitor of the Wnt signaling pathway which is involved in osteogenesis and, when inactivated, stimulates bone formation. To our knowledge, this effect has not been studied in the context of distraction osteogenesis (DO). Tibial DO was conducted on a total of 24 wild-type mice, which were then divided into 2 groups-a saline injection group (control) and an anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100 mg/kg intravenous injections of the antibody weekly until killing. The 12 mice in each group were subdivided into four time points according to post-osteotomy time of killing-11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation), with 3 mice per subgroup. After killing, the tibia specimens were collected for immunohistochemical analysis. Our results show that the group injected with anti-sclerostin had an earlier peak (day 11) in the distraction phase of the osteogenic molecules involved in the Wnt signaling pathway in comparison to the placebo group. In addition, downregulation of the inhibitors of this pathway was noted in the treatment group when compared with the placebo group. Furthermore, LRP-5 showed a significant increase in expression in the treatment group. Sclerostin inhibition has a significant effect on the DO process through its effect on the Wnt pathway. This effect was evident through the decreased effect of sclerostin on LRP-5 and earlier upregulation of the osteogenic molecules involved in this pathway.

Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy.

Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

Early health economic modelling of single-stage cartilage repair. Guiding implementation of technologies in regenerative medicine.

Both the complexity of clinically applied tissue engineering techniques for articular cartilage repair - such as autologous chondrocyte implantation (ACI) - plus increasing healthcare costs, and market competition, are forcing a shift in focus from two-stage to single-stage interventions that are more cost-effective. Early health economic models are expected to provide essential insight in the parameters driving the cost-effectiveness of new interventions before they are introduced into clinical practice. The present study estimated the likely incremental cost-effectiveness ratio (ICER) of a new investigator-driven single-stage procedure (IMPACT) compared with both microfracture and ACI, and identified those parameters that affect the cost-effectiveness. A decision tree with clinical health states was constructed. The ICER was calculated by dividing the incremental societal costs by the incremental Quality Adjusted Life Years (QALYs). Costs were determined from a societal perspective. A headroom analysis was performed to determine the maximum price of IMPACT compared with both ACI and microfracture, assuming a societal willingness to pay (WTP) of €30 000/QALY. One-way sensitivity analysis was performed to identify those parameters that drive the cost-effectiveness. The societal costs of IMPACT, ACI and microfracture were found to be €11 797, €29 741 and €6081, respectively. An 8% increase in all utilities after IMPACT changes the ICER of IMPACT vs. microfracture from €147 513/QALY to €28 588/QALY. Compared with ACI, IMPACT is less costly, which is largely attributable to the cell expansion procedure that has been rendered redundant. While microfracture can be considered the most cost-effective treatment option for smaller defects, a single-stage tissue engineering procedure can replace ACI to improve the cost-effectiveness for treating larger defects, especially if clinical non-inferiority can be achieved. Copyright © 2016 John Wiley & Sons, Ltd.

Near infrared spectroscopic assessment of developing engineered tissues: correlations with compositional and mechanical properties.

Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.

Ex vivo culture platform for assessment of cartilage repair treatment strategies.

There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.

Nondestructive Assessment of Engineered Cartilage Composition by Near Infrared Spectroscopy.

Tissue engineering presents a strategy to overcome the limitations of current tissue healing methods. Scaffolds, cells, external growth factors and mechanical input are combined in an effort to obtain constructs with properties that mimic native tissues. However, engineered constructs developed using similar culture environments can have very different matrix composition and biomechanical properties. Accordingly, a nondestructive technique to assess constructs during development such that appropriate compositional endpoints can be defined is desirable. Near infrared spectroscopy (NIRS) analysis is a modality being investigated to address the challenges associated with current evaluation techniques, which includes nondestructive compositional assessment. In the present study, cartilage tissue constructs were grown using chondrocytes seeded onto polyglycolic acid (PGA) scaffolds in similar environments in three separate tissue culture experiments and monitored using NIRS. Multivariate partial least squares (PLS) analysis models of NIR spectra were calculated and used to predict tissue composition, with biochemical assay information used as the reference data. Results showed that for combined data from all tissue culture experiments, PLS models were able to assess composition with significant correlations to reference values, including engineered cartilage water (at 5200 cm(-1), R = 0.68, p = 0.03), proteoglycan (at 4310 cm(-1), R = 0.82, p = 0.007), and collagen (at 4610 cm(-1), R = 0.84, p = 0.005). In addition, degradation of PGA was monitored using specific NIRS frequencies. These results demonstrate that NIR spectroscopy combined with multivariate analysis provides a nondestructive modality to assess engineered cartilage, which could provide information to determine the optimal time for tissue harvest for clinical applications.

Characteristic X-ray absorptiometry applied to the assessment of tissue-engineered cartilage development.

BACKGROUND: Transmission and tomographic X-ray measurements are useful in assessing bone structures, but only a few studies have examined cartilage growth because of the poor contrast in conventional X-ray imaging. OBJECTIVE: In this study, we attempted to use the linear attenuation coefficient (LAC) as a metric of tissue-engineered cartilage development, which would be useful in high-throughput screening of cartilage products. METHODS: Assuming that the LAC is related to the amount of extracellular matrix (ECM) in terms of the density and its atomic components, we measured X-ray absorption through tissue-engineered cartilage constructs. Characteristic X-ray beams from a molybdenum microfocus X-ray tube were employed to avoid beam hardening. The correlation of the LAC with mechanical properties was analyzed for verification. RESULTS: The LAC was higher for chondrocyte constructs and lower for fibroblast-dominant constructs and was consistent with the quantification of toluidine blue staining, which is a proof of ECM production. The LAC was positively correlated with the bending modulus but negatively correlated with the dynamic elastic modulus and stiffness, possibly because of the remaining scaffold. CONCLUSIONS: The LAC has the potential to be used as a metric of development of tissue-engineered cartilage. However, the calcified regions should be excluded from analysis to avoid decreasing the correlation between the LAC and the amount of ECM.

Enhanced depth-independent chondrocyte proliferation and phenotype maintenance in an ultrasound bioreactor and an assessment of ultrasound dampening in the scaffold.

Chondrocyte-seeded scaffolds were cultured in an ultrasound (US)-assisted bioreactor, which supplied the cells with acoustic energy around resonance frequencies (~5.0 MHz). Polyurethane-polycarbonate (BM), chitosan (CS) and chitosan-n-butanol (CSB) based scaffolds with varying porosities were chosen and the following US regimen was employed: 15 kPa and 60 kPa, 5 min per application and 6 applications per day for 21 days. Non-stimulated scaffolds served as control. For BM scaffolds, US stimulation significantly impacted cell proliferation and depth-independent cell population density compared to controls. The highest COL2A1/COL1A1 ratios and ACAN mRNA were noted on US-treated BM scaffolds compared to controls. A similar trend was noted on US-treated cell-seeded CS and CSB scaffolds, though COL2A1/COL1A1 ratios were significantly lower compared to BM scaffolds. Expression of Sox-9 was also elevated under US and paralleled the COL2A1/COL1A1 ratio. As an original contribution, a simplified mathematical model based on Biot theory was developed to understand the propagation of the incident US wave through the scaffolds and the model analysis was connected to cellular responses. Scaffold architecture influenced the distribution of US field, with the US field being the least attenuated in BM scaffolds, thus coupling more mechanical energy into cells, and leading to increased cellular activity.

Assessment of chondrogenic differentiation potential of autologous activated peripheral blood stem cells on human early osteoarthritic cancellous tibial bone scaffold.

INTRODUCTION: Current therapeutic regimens in osteoarthritis (OA) address mainly pain but not the slow progressive degradation of the extracellular matrix (ECM) and the loss of a chondrogenic phenotype in articular cartilage. In the present study, using an early OA cancellous bone scaffold, we aimed to uncover evidence of the successful hyaline cartilage regenerative capacity of autologous human granulocyte colony-stimulating factor (hG-CSF)-activated peripheral blood stem cells (AAPBSC) with growth factor addition. MATERIALS AND METHODS: AAPBSC were harvested in ten patients (median age 58 years, 8 females), and flow cytometry was performed for cell surface markers. Arthroscopically obtained cancellous bone scaffold specimens were seeded with AAPBSC. In Group 1, the scaffold was seeded with AAPBSC only, in Group 2, AAPBSC plus hyaluronic acid (HA), and in Group 3, AAPBSC plus HA, hG-CSF, and double-centrifuged platelet-rich plasma (PRP). The specimens were analyzed for cell attachment and proliferation by the fluorometric quantification of cellular DNA assay and scanning electron microscopy. Chondrogenic gene expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of Sox9, collagen type II (COL-2), and aggrecan. Histological sections of scaffold constructs for cartilaginous matrix formation were stained with toluidine blue (proteoglycan) and safranin O (sGAG) after 3 weeks. RESULTS: AAPBSC displayed especially high levels of CD29 and CD44 surface markers, as well as CD90, and CD105, while only a small proportion expressed CD34. Almost half of the seeded cells attached on the bone scaffolds in all three groups (not statistically significant), whereas the means of cell proliferation on day 7 compared to day 1 were statistically significant difference with the order of increase as group 3 > group 2 > group 1. RT-PCR showed statistically significant sequential increases in Sox9, COL-2, and Aggrecan all being highest in group 3. Histological analysis demonstrated cells in the cancellous bone scaffold with a round morphology, and ECM was positively stained by toluidine blue and safranin O indicating increased proteoglycan and glycosaminoglycan content, respectively, in the newly formed cartilage matrix. CONCLUSIONS: AAPBSC initiated chondrocyte differentiation on an autologous cancellous bone scaffold, and the addition of PRP and hG-CSF further stimulated cell proliferation toward a chondrocyte phenotype with potentiated Sox9 transcription resulting in sequential COL-2 and aggrecan mRNA increases that ultimately resulted in histologically confirmed increased proteoglycan and glucosaminoglycan content in newly formed hyaline cartilage.

Toward clinical application of tissue-engineered cartilage.

Since the late 1960s, surgeons and scientists envisioned use of tissue engineering to provide an alternative treatment for tissue and organ damage by combining biological and synthetic components in such a way that a long-lasting repair was established. In addition to the treatment, the patient would also benefit from reduced donor site morbidity and operation time as compared with the standard procedures. Tremendous efforts in basic research have been done since the late 1960s to better understand chondrocyte biology and cartilage maturation and to fulfill the growing need for tissue-engineered cartilage in reconstructive, trauma, and orthopedic surgery. Starting from the first successful generation of engineered cartilaginous tissue, scientists strived to improve the properties of the cartilaginous constructs by characterizing different cell sources, modifying the environmental factors influencing cell expansion and differentiation and applying physical stimuli to modulate the mechanical properties of the construct. All these efforts have finally led to a clinical phase I trial to show the safety and feasibility of using tissue-engineered cartilage in reconstructive facial surgery. However, to bring tissue engineering into routine clinical applications and commercialize tissue-engineered grafts, further research is necessary to achieve a cost-effective, standardized, safe, and regulatory compliant process.

Modeling chondrocyte patterns by elliptical cluster processes.

Superficial zone chondrocytes (CHs) of human joints are spatially organized in distinct horizontal patterns. Among other factors, the type of spatial CH organization within a given articular surface depends on whether the cartilage has been derived from an intact joint or the joint is affected by osteoarthritis (OA). Furthermore, specific variations of the type of spatial organization are associated with particular states of OA. This association may prove relevant for early disease recognition based on a quantitative structural characterization of CH patterns. Therefore, we present a point process model describing the distinct morphology of CH patterns within the articular surface of intact human cartilage. This reference model for intact CH organization can be seen as a first step towards a model-based statistical diagnostic tool. Model parameters are fitted to fluorescence microscopy data by a novel statistical methodology utilizing tools from cluster and principal component analysis. This way, the complex morphology of surface CH patters is represented by a relatively small number of model parameters. We validate the point process model by comparing biologically relevant structural characteristics between the fitted model and data derived from photomicrographs of the human articular surface using techniques from spatial statistics.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering.

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-ß1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-ß1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-ß1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.