Anatomical Tissue Engineering of the Anterior Cruciate Ligament Entheses.

The firm integration of anterior cruciate ligament (ACL) grafts into bones remains the most demanding challenge in ACL reconstruction, since graft loosening means graft failure. For a functional-tissue-engineered ACL substitute to be realized in future, robust bone attachment sites (entheses) have to be re-established. The latter comprise four tissue compartments (ligament, non-calcified and calcified fibrocartilage, separated by the tidemark, bone) forming a histological and biomechanical gradient at the attachment interface between the ACL and bone. The ACL enthesis is surrounded by the synovium and exposed to the intra-articular micromilieu. This review will picture and explain the peculiarities of these synovioentheseal complexes at the femoral and tibial attachment sites based on published data. Using this, emerging tissue engineering (TE) strategies addressing them will be discussed. Several material composites (e.g., polycaprolactone and silk fibroin) and manufacturing techniques (e.g., three-dimensional-/bio-printing, electrospinning, braiding and embroidering) have been applied to create zonal cell carriers (bi- or triphasic scaffolds) mimicking the ACL enthesis tissue gradients with appropriate topological parameters for zones. Functionalized or bioactive materials (e.g., collagen, tricalcium phosphate, hydroxyapatite and bioactive glass (BG)) or growth factors (e.g., bone morphogenetic proteins [BMP]-2) have been integrated to achieve the zone-dependent differentiation of precursor cells. However, the ACL entheses comprise individual (loading history) asymmetric and polar histoarchitectures. They result from the unique biomechanical microenvironment of overlapping tensile, compressive and shear forces involved in enthesis formation, maturation and maintenance. This review should provide a road map of key parameters to be considered in future in ACL interface TE approaches.

Osteochondral Interface: Regenerative Engineering and Challenges.

Osteochondral (OC) defects are debilitating for patients and represent a significant clinical problem for orthopedic surgeons as well as regenerative engineers due to their potential complications, which are likely to lead to osteoarthritis and related diseases. If they remain untreated or are treated suboptimally, OC lesions are known to impact the articular cartilage and the transition from cartilage to bone, that is, the cartilage-bone interface. An important component of the OC interface, that is, a selectively permeable membrane, the tidemark, still remains unaddressed in more than 90% of the published research in the past decade. This review focuses on the structure, composition, and function of the OC interface, regenerative engineering attempts with different scaffolding strategies and challenges ahead of us in recapitulating the native OC interface. There are different schools of thought regarding the structure of the native OC interface: stratified and graded. The former assumes the cartilage-to-bone interface to be hierarchically divided into distinct yet continuous zones of uncalcified cartilage-calcified cartilage-subchondral bone. The latter assumes the interface is continuously graded, that is, formed by an infinite number of layers. The cellular composition of the interface, either in respective layers or continuously changing in a graded manner, is chondrocytes, hypertrophic chondrocytes, and osteoblasts as moved from cartilage to bone. Functionally, the interface is assumed to play a role in enabling a smooth transition of loads exerted on the cartilage surface to the bone underneath. Regenerative engineering involves, first, a characterization of the native OC interface in terms of the composition, structure, and function, and, then, proposes the appropriate biomaterials, cells, and biomolecules either alone or in combination to eventually form a structure that mimics and functionally behaves similar to the native interface. The major challenge regarding regeneration of the OC interface appears to lie, in addition to others, in the formation of tidemark, which is a thin membrane separating the OC interface into two distinct zones: the avascular OC interface and the vascular OC interface. There is a significant amount of literature on regenerative approaches to the OC interface; however, only a small portion of them consider the importance of tidemark. Therefore, this review aims at highlighting the significance of the structural organization of the components of the OC interface and increasing the awareness of the orthopedics community regarding the importance of tidemark formation after clinical interventions or regenerative engineering attempts.

Roles of the calcified cartilage layer and its tissue engineering reconstruction in osteoarthritis treatment.

Sandwiched between articular cartilage and subchondral bone, the calcified cartilage layer (CCL) takes on both biomechanical and biochemical functions in joint development and ordinary activities. The formation of CCL is not only unique in articular cartilage but can also be found in the chondro-osseous junction adjacent to the growth plate during adolescence. The formation of CCL is an active process under both cellular regulation and intercellular communication. Abnormal alterations of CCL can be indications of degenerative diseases including osteoarthritis. Owing to the limited self-repair capability of articular cartilage and core status of CCL in microenvironment maintenance, tissue engineering reconstruction of CCL in damaged cartilage can be of great significance. This review focuses on possible tissue engineering reconstruction methods targeting CCL for further OA treatment.

Structural clues to articular calcified cartilage function: A descriptive review of this crucial interface tissue.

Articular calcified cartilage (ACC) has been dismissed, by some, as a remnant of endochondral ossification without functional relevance to joint articulation or weight-bearing. Recent research indicates that morphologic and metabolic ACC features may be important, reflecting knee joint osteoarthritis (OA) predisposition. ACC is less investigated than neighbouring joint tissues, with its component chondrocytes and mineralised matrix often being either ignored or integrated into analyses of hyaline articular cartilage and subchondral bone tissue respectively. Anatomical variation in ACC is recognised between species, individuals and age groups, but the selective pressures underlying this variation are unknown. Consequently, optimal ACC biomechanical features are also unknown as are any potential locomotory roles. This review collates descriptions of ACC anatomy and biology in health and disease, with a view to revealing its structure/function relationship and highlighting potential future research avenues. Mouse models of healthy and OA joint ageing have shown disparities in ACC load-induced deformations at the knee joint. This raises the hypothesis that ACC response to locomotor forces over time may influence, or even underlie, the bony and hyaline cartilage symptoms characteristic of OA. To effectively investigate the ACC, greater resolution of joint imaging and merging of hierarchical scale data will be required. An appreciation of OA as a 'whole joint disease' is expanding, as is the possibility that the ACC may be a key player in healthy ageing and in the transition to OA joint pathology.

Pathological, Morphometric and Correlation Analysis of the Modified Mankin Score, Tidemark Roughness and Calcified Cartilage Thickness in Rat Knee Osteoarthritis after Extracorporeal Shockwave Therapy.

The paper displayed the pathological changes and relationships of the modified Mankin score, tidemark roughness and calcified cartilage (CC) thickness by extracorporeal shockwave therapy (ESWT) (0.25 mJ/ mm2 with 800 impulses) on different positions of the medial and lateral rat knee OA joint. After the experiments, the articular cartilage was assessed using histomorphometry, image analysis and statistical method. In the micro-CT analysis, ESWT on medial groups were better than lateral groups in the trabecular volume and trabecular number. The data showed a strong negative correlation between the modified Mankin score and tidemark roughness (r = -0.941; P < 0.001). In terms of the relationship of tidemark roughness with CC thickness, the medial and Sham groups showed a significant negative correlation (r = -0.788, P = 0.022). Additionally, the Euclidean distance derived from 3D scatter plot analysis was an indicator of chondropathic conditions, exhibiting a strong correlation with OA stage in the articular cartilage of the femur (r = 0.911, P < 0.001) and tibia (r = 0.890, P < 0.001) after ESWT. Principle component analysis (PCA) further demonstrated that ESWT applied to medial locations had a better outcome than treatment at lateral locations for knee OA by comparing with Sham and OA groups, and CC thickness was the most important factor affecting hyaline cartilage repair after ESWT.

Primary Cilia Direct Murine Articular Cartilage Tidemark Patterning Through Hedgehog Signaling and Ambulatory Load.

Articular cartilage (AC) is essential for body movement but is highly susceptible to degenerative diseases and has poor self-repair capacity. To improve current subpar regenerative treatments, developmental mechanisms of AC should be clarified and, specifically, how its postnatal multizone organization is acquired. Primary cilia are cell surface organelles crucial for mammalian tissue morphogenesis. Although their importance for chondrocyte function is appreciated, their specific roles in postnatal AC morphogenesis remain unclear. To explore these mechanisms, we used a murine conditional loss-of-function approach (Ift88-flox) targeting joint-lineage progenitors (Gdf5Cre) and monitored postnatal knee AC development. Joint formation and growth up to juvenile stages were largely unaffected. However, mature AC (aged 2 months) exhibited disorganized extracellular matrix, decreased aggrecan and collagen II due to reduced gene expression (not increased catabolism), and marked reduction of AC modulus by 30%-50%. In addition, and unexpectedly, we discovered that tidemark patterning was severely disrupted, as was hedgehog signaling, and exhibited specificity based on regional load-bearing functions of AC. Interestingly, Prg4 expression was markedly increased in highly loaded sites in mutants. Together, our data provide evidence that primary cilia orchestrate postnatal AC morphogenesis including tidemark topography, zonal matrix composition, and ambulation load responses. © 2022 American Society for Bone and Mineral Research (ASBMR).

Thickness determination of the tidemark of human articular cartilage using high-resolution micro-XRF imaging of zinc and lead.

Objective: The objective of the study was to specify the thickness of Zn and Pb accumulation within the tidemark (TM), a narrow structure between the non-calcified and the calcified articular cartilage. It is considered an active or resting calcification front. This banded structure of the cartilage-bone interface is known to undergo changes in osteoarthritis. Therefore, gaining knowledge about this structure is of interest. Methods: Femoral head samples were collected from patients suffering from various bone diseases, 6 samples have been investigated. Thin bone slices (3 â€‹µm thick) were measured with high resolution synchrotron micro-X-ray fluorescence (SR micro-XRF) analysis using a beam with dimensions of 500 â€‹× â€‹800 â€‹nm2. The tidemark region was found in all analyzed samples. The Savitzky-Golay filter was used to smooth the measured imaging data and Kaplan-Meier estimation to gain reliable tidemarks medians for Pb and Zn. To our knowledge this was the first time that these methods have been applied to gain information on histological structures obtained by elemental imaging. Results: The thickness of the Zn and Pb layer ranged from about 3 to 11 â€‹µm for Zn and 4-14.5 â€‹µm for Pb. Our Zn ratios (TM/matrix) were found to be 1.5-3-fold ratio between Zn tidemark values and in mineralized matrix and are similar in all samples. Conclusions: The determined thickness of the layer is much smaller than found in previous measurements with the beam having 20 â€‹× â€‹14 â€‹µm2 size. The Zn ratios agree with our previous findings.

Nanoindentation: An advanced procedure to investigate osteochondral engineered tissues.

Osteochondral scaffolds are emerging as a promising alternative for articular cartilage regeneration, although with still controversial results. In particular, the restoration of the osteochondral interface remains an open challenge. The current available investigative procedures are not optimal to quantify the properties of this region, neither to evaluate the quality of the regenerated tissue with respect to the physiological one. This study investigates an advanced procedure able to quantitatively evaluate the mechanical gradient between stiff and compliant tissues, such as in the osteochondral region where the interface between hyaline and calcified cartilage (tidemark) plays an integral role in transferring articular loads from the compliant articular surface to the stiffer underlying bone. A series of nanoindentation line scans was performed along the tidemark - starting from hyaline and expanding across calcified cartilage - on histological sections derived from sheep osteochondral tissue regenerated by a three-layered biomimetic scaffold, as well as to the adjacent healthy tissue for comparative purposes. After an accurate assessment of the indentation parameters, a sigmoid curve-fit function was applied on the reduced modulus profiles to extract gap, width and regularity of the mechanical transition. The designed procedure succeeded in quantitatively assessing the transition between compliant and stiff regions, limiting experimental issues that generally affect the reliability of the indentation mechanical data, such as apex-blunt indenter tip effect, surface roughness, and influence of the substrate. Among the evaluated parameters, the mechanical gap highlighted the main difference between native and regenerated tissues. Thanks to the information retrievable through this procedure, this load transmission area can be further investigated, providing data to tailor osteochondral engineered tissues in the future.

3D morphometric analysis of calcified cartilage properties using micro-computed tomography.

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (µCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a µ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

High-Frequency Ultrasound Imaging of Tidemark In Vitro in Advanced Knee Osteoarthritis.

High-frequency ultrasound imaging has been widely adopted for assessment of the degenerative changes of articular cartilage in osteoarthritis (OA). Yet, there are few reports on investigating its capability to evaluate subchondral bone. Here, we employed high-frequency ultrasound imaging (25 MHz) to examine in vitro the tidemark in cylindrical osteochondral disks (n = 33) harvested from advanced OA knees of humans. We found good correspondence in morphology observed by ultrasound imaging and micro-computed tomography. Ultrasound roughness index (URI) of tidemark was derived from the raw radiofrequency signals to compare with bone quality factors, including bone volume fraction (BV/TV) and bone mineral density (BMD) measured by micro-computed tomography, using the Spearman correlation (ρ). URI of the tidemark was negatively associated with the subchondral plate BV/TV (ρ = -0.73, p <0.001), BMD (ρ = -0.40, p = 0.020), as well as the underneath trabecular bone BV/TV (ρ = -0.39, p = 0.025) and BMD (ρ = -0.43, p = 0.012). In conclusion, this preliminary study demonstrated that morphology measured by high-frequency ultrasound imaging could reflect the quality of the subchondral bone. High-frequency ultrasound is a promising imaging tool to evaluate the changes of the subchondral bone in addition to those of the overlying cartilage in OA.

On the heterogeneity of the femoral enthesis of the human ACL: microscopic anatomy and clinical implications.

BACKGROUND: Most ruptures of the native anterior cruciate ligament (ACL) and ACL graft occur at, or near, the femoral enthesis, with the posterolateral fibers of the native ligament being especially vulnerable during pivot landings. Characterizing the anatomy of the ACL femoral enthesis may help us explain injury patterns which, in turn, could help guide injury prevention efforts. It may also lead to improved anatomic reconstruction techniques given that the goal of such techniques is to replicate the knee's normal anatomy. Hence, the aim of this study was to investigate the microscopic anatomy of the ACL femoral enthesis and determine whether regional differences exist. METHODS: Fifteen human ACL femoral entheses were histochemically processed and sectioned along the longitudinal axis of the ACL at 20, 40, 60, and 80 % of the width of the enthesis. Four thick sections (100 µm) per enthesis were prepared, stained, and digitized. From these sections, regional variations in the quantity of calcified and uncalcified fibrocartilage, the angle at which the ligament originates from the bone, and the shape profile of the tidemark were quantified. RESULTS: At least 33 % more calcified fibrocartilage and 143 % more uncalcified fibrocartilage were found in the antero-inferior region, which corresponds to the inferior margin of the origin of the anteromedial ACL fibers, than all other regions (Ps < 0.05). In addition, the anteromedial fibers of the ACL originated from the femur at an angle six times greater than did its posterolateral fibers (P = 0.032). Finally, average entheseal tidemark profiles correlated bilaterally (Pearson's r = 0.79; P = 0.036), the most common profile being convex with a single re-entrant. CONCLUSIONS: Systematic regional differences were found in fibrocartilage quantity and collagen fiber attachment angles. The marked differences may reflect differences in the loading history of the various regions of the ACL femoral enthesis. These differences, which could affect the potential for injury, should also be considered when developing new ACL reconstruction approaches.

Osteochondral autografts.

The healing potential for articular cartilage lesions is limited due to many physiological, local and mechanical factors. Spontaneous healing of partial- and full-thickness lesions is slow, and subsequent tissue response is usually not durable. In symptomatic, and high-demand, patients, a definitive treatment modality must be offered which allows for a sustained recovery with minimal debilitation. Injuries, which damage the subchondral bone, heal with the formation of fibrocartilage. This tissue fails long-term survival because of its inability to withstand the variable cyclic loads and compression forces that it is subjected to. While regeneration of the damaged cartilage by an entirely new articular surface is a goal beyond current available techniques, repair of the osteochondral defects with normal hyaline cartilage is possible by various options. Osteochondral defects that are larger then 2 cm are best treated by osteochondral autograft technique. The short-term outcomes of the present series show excellent results.

Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

BACKGROUND: Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. PURPOSE/HYPOTHESIS: The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. STUDY DESIGN: Descriptive laboratory study. METHODS: After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. RESULTS: Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and fibrocartilage had the same mean Col2 percentage stain; however, fibrocartilage was distinguished by heavy Col1 deposits in the deep zone, a 2-fold higher mean Col1 percentage stain (P = .001), and lower surface integrity (P = .03). ZCT scores correlated with GAG content and the ICRS-II overall assessment score, especially when combined with the PLM score for collagen organization (R = 0.82). Histological scores of the deep zone strongly predicted the ICRS-II overall assessment score (R = 0.99). CONCLUSION: The ICRS-II overall repair assessment score and GAG content correlated with the extent of Col2 deposition free of fibrosis in the deep/middle zone rather than bulk accumulation of Col2. CLINICAL RELEVANCE: Biopsy tissue from the BST-CarGel randomized clinical trial (microfracture without and with BST-CarGel, as treatment groups were not unblinded) showed regenerated tissue consistent with a chondroinduction mechanism in at least half of the treated lesions.

Deleterious effects of osteoarthritis on the structure and function of the meniscal enthesis.

OBJECTIVE: The ability of menisci to prevent osteoarthritis (OA) is dependent on the integrity of the complex meniscal entheses, the attachments of the menisci to the underlying subchondral bone (SB). The goal of this study was to determine mechanical and structural changes in meniscal entheses after the onset of OA. DESIGN: Healthy and osteoarthritic meniscal entheses were evaluated for changes in histomorphological characteristics, mineralization, and mechanical properties. Glycosaminoglycans (GAG) and calcium in the insertion were evaluated with histological staining techniques. The extent of calcium deposition was assessed and tidemark (TM) integrity was quantified. Changes in the mineralized zone of the insertion were examined using micro-computed tomography (µCT) to determine bone mineral density, cortical zone thickness, and mineralization gradient. Mechanical properties of the entheses were measured using nano-indentation techniques to obtain material properties based on viscoelastic analysis. RESULTS: GAG thickness in the calcified fibrocartilage (CFC) zone and calcium content were significantly greater in osteoarthritic anterior meniscal entheses. TM integrity was significantly decreased in OA tissue, particularly in the medial anterior (MA) enthesis. The mineralized zone of osteoarthritic meniscal entheses was significantly thicker than in healthy entheses and showed decreased bone mineral density. Fitting of mineralization data to a sigmoidal Gompertz function revealed a lower rate of increase in mineralization in osteoarthritic tissue. Analysis of viscoelastic mechanical properties revealed increased compliance in osteoarthritic tissue. CONCLUSIONS: These data suggest that significant changes occur at meniscal enthesis sites with the onset of OA. Mechanical and structural changes in meniscal entheses may contribute to meniscal extrusion, which has been shown to increase the progression of OA.

Differential accumulation of lead and zinc in double-tidemarks of articular cartilage.

INTRODUCTION: Long-term exposure to increased lead (Pb) concentrations is associated with several chronic diseases. The divalent cation zinc (Zn) is essential for numerous enzymes. In a recent study we found remarkably elevated concentrations of Pb and Zn in the tidemark (TM), which is the mineralization front of human articular cartilage. OBJECTIVE: Duplication or multiplication of TMs occurs with advancing age or degeneration. We hypothesized that trace elements accumulate in TMs as a function of time. Thus, in cases of double TMs, the deep (older) TM should contain higher Pb and Zn concentrations than the superficial (younger) TM. DESIGN: Undecalcified tissue from articular cartilage and subchondral bone of femoral heads and patellae was examined by synchrotron radiation induced confocal micro X-ray fluorescence analysis and by quantitative backscattered electron imaging to determine the local distribution of Ca, Zn, and Pb in this tissue. RESULTS: The evaluation of X-ray fluorescence intensities in double TMs revealed in average a 2.6-fold higher Pb level in the deep TM compared to the superficial TM while Zn concentrations were similar. Pb and Zn contents were significantly enhanced in the deep TM (Pb: 35-fold, Zn: five-fold) and in the superficial TM (Pb: 12-fold, Zn: five-fold) compared to the bone level. CONCLUSION: For the first time a differential accumulation of Pb and Zn is documented in regions with double TMs revealing various timescales for the accumulation of these elements. Increased amounts of Pb are present in the TMs (up to the 62-fold of the bone level) featuring a potential source of internal Pb release if the TM region is destroyed.

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.

Proceso biológico del desarrollo epifisario:: aparición y formación del centro secundario de osificación / Processus biologique du développement épiphysaire:: survenue et formation du point d'ossification secondaire / Biological process of epiphyseal development:: appearance and formation of secondary center of ossification

Los huesos se constituyen de tejido conectivo especializado que conforma el principal órgano de soporte del cuerpo. Las epífisis de los huesos largos experimentan durante el período perinatal diversos cambios estructurales que conllevan a la formación del centro secundario de osificación (CSO), responsable del crecimiento esférico de la epífisis. En este artículo se exponen los cambios biológicos característicos de las diversas etapas del desarrollo del CSO, mediante una descripción detallada de los eventos histológicos que ocurren en cada etapa e ilustrando la coordinación de procesos y el patrón de desarrollo epifisario. Por consiguiente, este artículo facilitará la comprensión de dichos procesos, la creación de modelos matemáticos de los diferentes eventos y proporcionará una base teórica para trabajos futuros que investiguen el desarrollo y crecimiento epifisario(AU)

Bones are the specialized connective tissue shaping the main support organ of the body. Long bone epiphysis underwent many structural changes during the perinatal period leading to formation of a secondary center of ossification (SCO), accounting for the epiphyseal spherical growth. In present paper are exposed the characteristic biological changes of the different SCO development stages thorough a detailed description of histological events occurring in each stage and illustrating the processes coordination and the epiphyseal development pattern. Therefore, present article will allows the understanding of such processes, creation of mathematical models of different events, as well as a theoretical basis for future papers researching the epiphyseal development and growth(AU)

Les os sont constitués de tissu connectif spécialisé formant l'organe de support principal du corps. Les épiphyses des os longs subissent plusieurs changements de structure lors de la période périnatale impliquant la formation du point d'ossification sécondaire (POS), responsable de la croissance sphérique de l'épiphyse. Dans cet article, les changements biologiques caractéristiques des diverses étapes du développement du POS sont exposés par une description détaillée des évènements histologiques se produissant dans chaque étape, et en montrant la coordination des processus et le modèle de développement épiphysaire. Cet article va donc faciliter la compréhension de ces dits processus, la création de modèles mathématiques des différents évènements, et donner une base théorique pour des travaux futurs étudiant le développement et la croissance épiphysaires(AU)