Pathologist Computer-Aided Diagnostic Scoring of Tumor Cell Fraction: A Swiss National Study.

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems.

No effect of subperiosteal growth factor application on periosteal neo-chondrogenesis in osteoperiosteal bone grafts for osteochondral defect repair.

PURPOSE: The purpose of this study was to examine the effect of subperiosteal injection of chondroinductive growth factors on the histological and biomechanical outcome of autologous osteoperiosteal grafts. METHODS: Thirty six standardised osteochondral defects were created in the trochlear groove of 18 Göttinger Minipigs and evaluated after six, 12 and 52 weeks. Defects were treated with press-fit implantation of autologous osteoperiosteal cylindrical block-grafts with or without subperiosteal injection of a chondroinductive growth factor mixture (GFM). RESULTS: Histomorphological analysis showed complete osseointegration of all grafts from six weeks. The periosteum remained in place in 35 of 36 cases. Fibrocartilagineous repair tissue formation occurred at the cambium layer with a maximum at 12 weeks in both groups. Histomorphological grading and biomechanical testing showed highest values at 12 weeks, with signs of tissue degradation at one year. There was no significant difference between both groups. CONCLUSION: Transplantation of autologous osteoperiosteal grafts is an effective method to restore subchondral bone defects, but not the overlying cartilage as the repair tissue deteriorates in the long term. Subperiosteal growth factors injection did not stimulate tissue differentiation on a biomechanical and histomorphological level.

Increasing Concentration of Sinovial: Effect on Cartilage Protection in a Rabbit ACLT Model.

OBJECTIVE: This study aimed to test the hypothesis that administration of increasing doses of Sinovial (hyaluronic acid [HA]), would exhibit a dose-dependent effect on the prevention of cartilage degradation, without local and systemic toxicity. METHODS: Twenty-seven adult rabbits were subjected to anterior cruciate ligament transection (ACLT). Two Sinovial products containing HA concentrations of 1.6% and 2.4% were used as active treatment, and 0.9% saline was used as control and injected intra-articularly 7 days post ACLT. Radiographs were taken prior to surgery, at injection and sacrifice times. After euthanasia, 8 weeks postsurgery, knee joints were observed macroscopically using India ink staining with OARSI (Osteoarthritis Research Society International) scoring and histologically using modified Mankin scoring. The synovial membranes were analyzed using Cake classification. RESULTS: No intraoperative complications were observed. One week postinjection, 4 animals in the HA 2.4% group developed subcutaneous nodules that disappeared spontaneously. No inflammation of the synovial membrane was ever observed. The control group exhibited the maximum uptake of India ink 2.22 ± 0.14. HA groups exhibited a dose-dependent (P = 0.02) reduction in India ink uptake: 1.75 ± 0.17 for HA 1.6% and 1.58 ± 0.14 for HA 2.4%. The most marked dose-dependent effect of this study was a reduction of modified Mankin score for HA groups, with the 2.4% treatment achieving a statistically significant improvement as compared with the control group (7.19 ± 0.85 for saline, 4.65 ± 0.66 for HA 1.6%, and 3.53 ± 0.59 for HA 2.4%; P = 0.005). CONCLUSIONS: A dose-dependent protective effect on cartilage was observed after injection of both HA solutions.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes.

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma.

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

Evaluation of early tissue reactions after lumbar intertransverse process fusion using CT in a rabbit.

OBJECTIVE: The objective of the study was to evaluate tissue reactions such as bone genesis, cartilage genesis and graft materials in the early phase of lumbar intertransverse process fusion in a rabbit model using computed tomography (CT) imaging with CT intensity (Hounsfield units) measurement, and to compare these data with histological results. MATERIALS AND METHODS: Lumbar intertransverse process fusion was performed on 18 rabbits. Four graft materials were used: autograft bone (n = 3); collagen membrane soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) (n = 5); granular calcium phosphate (n = 5); and granular calcium phosphate coated with rhBMP-2 (n = 5). All rabbits were euthanized 3 weeks post-operatively and lumbar spines were removed for CT imaging and histological examination. RESULTS: Computed tomography imaging demonstrated that each fusion mass component had the appropriate CT intensity range. CT also showed the different distributions and intensities of bone genesis in the fusion masses between the groups. Each component of tissue reactions was identified successfully on CT images using the CT intensity difference. Using CT color mapping, these observations could be easily visualized, and the results correlated well with histological findings. CONCLUSIONS: The use of CT intensity is an effective approach for observing and comparing early tissue reactions such as newly synthesized bone, newly synthesized cartilage, and graft materials after lumbar intertransverse process fusion in a rabbit model.

Multilineage differentiation potential of equine blood-derived fibroblast-like cells.

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Efficacy of HYADD®4-G single intra-discal injections in a rabbit model of intervertebral disc degeneration.

BACKGROUND: Various biomaterials/technologies have been tested for treatment of intervertebral disc (IVD) degeneration (IDD). Only few non-surgical options exist. OBJECTIVE: Assessment of efficacy and safety of the hyaluronic acid derivative hydrogel HYADD®4-G in IDD using a well-established rabbit annular puncture model. METHODS: Rabbits were punctured at two IVDs to induce IDD. Thirty days after, IVDs were injected with HYADD®4-G or saline. IVD hydration, height, appearance and tissue organization were assessed by radiographs, MRI and histopathology. Safety of HYADD®4-G injection was evaluated in non-punctured IVDs. RESULTS: HYADD®4-G injection restored disc height to over 75% of the pre-punctured disc, saline injections led to 50% of initial disc height. Compared to saline, HYADD®4-G treatment resulted in improved water retention as revealed by MRI quantification. 83.3% of HYADD®4-G injected discs had normal appearance and reached grade I of the Pfirrmann scale. Regarding tissue organization and cellularity, HYADD®4-G treatment resulted in significantly lower IDD scores than saline (p < 0.01). HYADD®4-G injected into healthy IVDs did not induce inflammation or foreign body reactions. CONCLUSIONS: Intra-discal HYADD®4-G injection is safe and has therapeutic benefits: IDD could be limited through restoration of disc height and hydration and maintenance of normal IVD tissue organization.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells.

In this study, a time-course comparison of human articular chondrocytes (HAC) and bone marrow-derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14-/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1-2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3-4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index.

Cartilage tissue engineering for degenerative joint disease.

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects.

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

Visual histological grading system for the evaluation of in vitro-generated neocartilage.

Here we present the development of a visual evaluation system for routine assessment of in vitro-engineered cartilaginous tissue. Neocartilage was produced by culturing human articular chondrocytes in pellet culture systems or in a scaffold-free bioreactor system. All engineered tissues were embedded in paraffin and were sectioned and stained with Safranin O-fast green. The evaluation of each sample was broken into 3 categories (uniformity and intensity of Safranin O stain, distance between cells/amount of matrix produced, and cell morphology), and each category had 4 components with a score ranging from 0 to 3. Three observers evaluated each sample, and the new system was independently tested against an objective computer-based histomorphometry system. Pellets were also assessed biochemically for glycosaminoglycan (GAG) content. Pellet histology scores correlated significantly with GAG contents and were in agreement with the computer-based histomorphometry system. This system allows a valid and rapid assessment of in vitro-generated cartilaginous tissue that has a relevant association with objective parameters indicative of cartilage quality.

Monopolar radiofrequency treatment of partial-thickness cartilage defects in the sheep knee joint leads to extended cartilage injury.

BACKGROUND: The application of radiofrequency energy to smooth and stabilize the cartilage surface has become increasingly controversial. There is little knowledge on extended-term effects, such as cartilage viability. PURPOSE: To analyze the effect of radiofrequency treatment on artificially created partial-thickness defects in the femoral cartilage of sheep knee joints 24 weeks after surgery. STUDY DESIGN: Controlled laboratory study. METHODS: Grade II cartilage surface defects on the medial and lateral femoral condyles were artificially created in sheep for in vivo analysis. The cartilage lesions were treated alternately on the lateral or the medial condyle using a monopolar radiofrequency probe. Radiofrequency treatment was performed in a freehand technique until surface smoothing without change of cartilage color was seen. At 24 weeks after surgery, cartilage samples were harvested and were processed for macroscopic and histological evaluation. To analyze the effect of radiofrequency at time zero, samples of sheep femoral condyle cartilage with and without artificially created clefts were treated in vitro with radiofrequency. Evaluation was performed by scanning electron and confocal microscopy. RESULTS: At 24 weeks after surgery, grade IV cartilage defects were detected in all radiofrequency-treated samples. The histological findings showed a central ulcer and dead chondrocytes in the radiofrequency-treated regions. The radiofrequency-treated cartilage revealed partial surface irregularities with partial-defect repair. After radiofrequency treatment in vitro, samples at time zero showed smoothing of the artificially created clefts, as seen by scanning electron microscopy. Confocal microscopy showed necrosis of chondrocytes over approximately one fourth of the upper cartilage thickness. CONCLUSION: Even if chondrocyte death is seen only in approximately one fourth of the upper cartilage layers in the sheep femur after in vitro application, radiofrequency treatment can cause damage to cartilage 24 weeks after application. CLINICAL RELEVANCE: Caution is recommended in the application of monopolar radiofrequency energy by visual control to partial-thickness cartilage defects. Irregular fronds of chondromalacia may be unattractive but represent viable articular cartilage. Using radiofrequency to obtain a more visually pleasing smooth surface may be counterproductive.

The Role of Cells in Meniscal Guided Tissue Regeneration: A Proof of Concept Study in a Goat Model.

OBJECTIVE: Successful repair of defects in the avascular zone of meniscus remains a challenge in orthopedics. This proof of concept study aimed to investigate a guided tissue regeneration approach for treatment of tears in meniscus avascular zone in a goat model. DESIGN: Full-depth longitudinal tear was created in the avascular zone of the meniscus and sutured. In the two treatment groups, porcine collagen membrane was wrapped around the tear without (CM) or with injection of expanded autologous chondrocytes (CM+cells), whereas in the control group the tear remained only sutured. Gait recovery was evaluated during the entire follow-up period. On explantation at 3 and 6 months, macroscopic gross inspection assessed healing of tears, degradation of collagen membrane, potential signs of inflammation, and osteoarthritic changes. Microscopic histology scoring criteria were developed to evaluate healing of tears, the cellular response, and the inflammatory response. RESULTS: Gait recovery suggested protective effect of collagen membrane and was supported by macroscopical evaluation where improved tear healing was noted in both treated groups. Histology scoring in CM compared to suture group revealed an increase in tear margins contact, newly formed connective tissue between margins, and cell formations surrounded with new matrix after 3 months yet not maintained after 6 months. In contrast, in the CM+cells group these features were observed after 3 and 6 months. CONCLUSIONS: A transient, short-term guided tissue regeneration of avascular meniscal tears occurred upon application of collagen membrane, whereas addition of expanded autologous chondrocytes supported more sustainable longer term tear healing.

Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C.

Enchondromatosis (Ollier disease, Maffucci syndrome) is a rare developmental disorder characterized by multiple enchondromas. Not much is known about its molecular genetic background. Recently, an activating mutation in the parathyroid hormone receptor type 1 (PTHR1) gene, c.448C>T (p.R150C), was reported in two of six patients with enchondromatosis. The mutation is thought to result in upregulation of the IHH/PTHrP pathway. This is in contrast to previous studies, showing downregulation of this pathway in other cartilaginous tumors. Therefore, we investigated PTHR1 in enchondromas and chondrosarcomas from 31 enchondromatosis patients from three different European countries, thereby excluding a population bias. PTHR1 protein expression was studied using immunohistochemistry, revealing normal expression. The presence of the described PTHR1 mutation was analyzed, using allele-specific oligonucleotide hybridization confirmed by sequence analysis, in tumors from 26 patients. In addition, 11 patients were screened for other mutations in the PTHR1 gene by sequence analysis. Using both allele-specific oligonucleotide hybridization and sequencing, we could neither confirm the previously found mutation nor find any other mutations in the PTHR1 gene. These results indicate that the PTHR1 gene is not, in contrast to previous suggestions, the culprit for enchondromatosis.

Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study.

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-d,l-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.

Adult human neural crest-derived cells for articular cartilage repair.

In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile--a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC-based engineered tissues for the treatment of traumatic articular cartilage lesions.

Efficacy Evaluation of a New Hyaluronan Derivative HYADD® 4-G to Maintain Cartilage Integrity in a Rabbit Model of Osteoarthritis.

Objective: To test the efficacy of a hyaluronan derivative (HYADD®4-G) in a model of osteoarthritis (anterior cruciate ligament [ACLT]) and to compare its efficacy with the injection of growth factors. Design: In a first experimental set-up, specially selected for treatment scheme with published studies on hyaluronan or growth factor efficacy in osteoarthritis, saline, HYADD®4-G, rh-BMP-7, and the treatments of rh-BMP-7 or rh-BMP-2 with HYADD®4-G were injected after ACLT, for five times starting 3 weeks after ACLT. Euthanasia was at day 70. The knees were evaluated by gross morphological observation, x-ray, and histology (Study A). In a second experimental set-up selected to evaluate the efficacy of three viscosupplement injections, starting 4 weeks after ACTL, HYADD®4-G was compared to saline (Study B). Results: (A) X-ray analysis showed more damage in the saline group than all other treatment groups (2.67 ± 0.61 for saline, 0.83 ± 0.26 for HYADD®4-G, 1.67 ± 0.82 for HYADD®4-G with rh-BMP-2, 0.75 ± 0.76 for HYADD®4-G with rh-BMP-7, and 1.58 ± 0.49 for rh-BMP-7), P < 0.05. In the femoral condyle, the Mankin's score for HYADD®4-G with rh-BMP-2, HYADD®4-G with rh-BMP-7, and rh-BMP7 alone was statistically lower compared to saline in the medial part; in the lateral part a significant lower value was observed in the HYADD®4-G with the rh-BMP-2 group. (B) The Kellgren and Lawrence score and Mankin's score was lower in the HYADD®4-G group than in the saline group (P < 0.002 and P = 0.0031). Conclusions: These two studies suggest that HYADD®4-G delayed the cartilage degeneration and that the association of HYADD®4-G with growth factors is synergistic.

International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

Preclinical Studies for Cartilage Repair: Recommendations from the International Cartilage Repair Society.

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.