Arthroscopic gel-type autologous chondrocyte implantation presents histologic evidence of regenerating hyaline-like cartilage in the knee with articular cartilage defect.

PURPOSE: To investigate the clinical, radiological, and histological results of arthroscopic gel-type autologous chondrocyte implantation (GACI) in treating chondral defects of the knee. METHODS: This study prospectively examined five males and five females with a mean age of 40.3 ± 10.3 years who underwent arthroscopic GACI between March 2012 and February 2013. The gel comprised a mixture of 1 ml of fibrinogen plus 0.1-0.2 ml of thrombin. The mean size of chondral defect was 2.9 ± 1.2 cm2 (range 1.2-5.4 cm2). International knee documentation committee (IKDC) subjective score, knee injury and osteoarthritis outcome score (KOOS), knee society score, and visual analog scale (VAS) for pain were assessed preoperatively and during regular follow-up examinations performed for up to 5 years postoperatively. Serial magnetic resonance imaging was performed for up to 2 years after the surgery to observe healing, using the modified magnetic resonance observation of cartilage repair tissue (MOCART) score. In eight patients, second-look arthroscopy was performed at 1 year after the implantation to assess the status of treated cartilage, and a portion of regenerated cartilage was harvested for histologic evaluation. RESULTS: The mean VAS score (p = 0.045), IKDC subjective score (p = 0.041), KOOS pain (p = 0.025), KOOS activities of daily living (p = 0.048), and KOOS quality of life (p = 0.029) showed significant improvement at 5 years after the surgery. The modified MOCART evaluation showed that the scores were 59.5 ± 29.4 and 85.0 ± 8.0 at 12 weeks and 2 years after the operation, respectively. Histologic examination demonstrated a mean regenerated cartilage thickness of 3.5 ± 0.8 mm and a mean Oswestry score of 8.2 ± 1.8. Immunohistochemistry analysis showed that the expression of collagen type II was more evident and more evenly distributed than collagen type I in regenerated cartilage. There was a significant correlation between Oswestry score and change in VAS scale from postoperative 2-5 years. CONCLUSIONS: Arthroscopic GACI produces satisfactory clinical and radiologic outcomes, and histologic evaluation confirms sufficient regeneration of hyaline-like cartilage that correlates with improved symptoms. Therefore, it is an acceptable, minimally invasive, and technically simple option for the restoration of cartilage defects of the knee. LEVEL OF EVIDENCE: IV.

Histological analysis of hyalinised keloidal collagen formation in earlobe keloids over time: collagen hyalinisation starts in the perivascular area.

Keloids grow and do not regress. They are characterised histologically by hyalinised keloidal collagen (HKC). HKC amounts vary, and the mechanism by which they form is unclear. To clarify how HKCs form and whether their formation associates with specific clinical features, we studied the histological findings of earlobe keloids and compared them with respective clinical features. A total of 50 earlobe keloids from 43 patients were used for histological analysis of keloid size (mm2 ), HKC area (mm2 ) and HKC area ratio (%). As a result, keloid durations ranged from 3 months to >13 years. Early-stage keloids exhibited little HKC and a tendency for the HKCs to locate in perivascular regions. In later-stage keloids, the HKCs were extremely interconnected and formed a thick bitten donut-shaped region. HKC area ratios correlated positively with keloid duration (r2 = 0·58, P<0·05). HKC area ratios and keloid durations did not correlate with keloid sizes. These patterns of HKC formation and growth may explain why local therapies, which effectively remove fibroblasts and accumulated collagen but not HKCs, are ineffective in older keloids. Keloids should be promptly treated after diagnosis, and older keloids with extensive HKCs may require surgical excision followed by radiotherapy.

[Effects of in vitro continuous passaging on the phenotype of mouse hyaline chondrocytes and the balance of the extra- cellular matrix].

OBJECTIVE: This study aimed to investigate the effects of in vitro continuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix (ECM). METHODS: Enzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passages in vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the mRNA changes in the marker genes, routine genes, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities. RESULTS: After continuous expansion in vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased (P < 0.05), and it was almost negatively expressed by P5 chondrocytes. By contrast, the down regulation of routine genes was insignificant. The gene expression levels of MMPs and TIMPs both decreased (P < 0.05), but the change in MMP-1 and TIMP-1 was not statistically significant (P > 0.05). Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes (P < 0.05). CONCLUSION: Serially passaged chondrocytes dedifferentiated and lost specific phenotypic characteristics during in vitro expansion culture. Simultaneously, the anabolism and catabolism of the cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0-P2 chondrocytes should be used.

Effect of Enoxaparin Sodium on Experimentally-Induced Myringosclerosis in Rats.

OBJECTIVE: To evaluate the effectiveness of enoxaparin sodium (ES) on experimentally-induced myringosclerosis in rats. MATERIALS AND METHODS: Twenty Wistar albino-type rats weighing up to 250-300 g each were randomized into four groups containing five rats each and were then bilaterally myringotomized. The control group (n=5) received intratympanic serum physiologic injections, whereas ES2 (n=5), ES4 (n=5), and ES6 groups (n=5) received intratympanic ES of 2000 IU, 4000 IU, and 6000 IU, respectively, for 10 days after myringotomy. Rats were sacrificed at 60 days after intratympanic application and were then prepared for histopathologic evaluation. RESULTS: As for tympanic membrane hyaline degeneration, there were statistically significant differences among the control, ES2, ES4, and ES6 groups (p<0.05). As for fibrosis formation on tympanic membranes, a statistically significant difference was observed among the control and study groups; however, although not statistically significant, the formation of fibrosis was slowed down in the ES2 and ES4 groups compared with the control group. The control and study groups did not show any significant difference for calcification, hyperemia, and tympanic membrane thickening (p>0.05). CONCLUSION: Although our study and control groups comprised limited number of animals, and only one parameter demonstrated a statistically significant difference between the groups, ES may have an ameliorating effect on myringosclerosis induced by myringotomy in the tympanic membranes of rats. ES proved to be effective in the prevention of hyaline disc formation. Further studies should be conducted for better understanding of the effects of low-molecular-weight heparin (LMWH) (i.e., enoxaparin) on myringosclerosis.

Force distribution is more important than its intensity!

A common question about root resorption is raised in orthodontic practice: What is more important, the intensity of force or its distribution along the root, periodontal and alveolar structures? Diffuse distribution of forces applied to periodontal tissues during tooth movement tends not to promote neither extensive areas of cell matrix hyalinization nor significant death of cementoblasts that lead to root resorption. However, focal distribution or concentration of forces within a restricted area--as it occurs in tipping movements, even with forces of lower intensity--tend to induce extensive areas of hyalinization and focal death of cementoblasts, which is commonly associated with root resorption. In tipping movements, the apical regions tend to concentrate more forces, in addition to wounding the cementoblasts due to the smaller dimension of their root structure as well as their cone shape. For this reason, there is an increase in root resorption. In the cervical region, on the other hand, the large area resulting from a large diameter and bone crown deflection tends to reduce the effects of forces, even when they are more concentrated, thus rarely inducing death of cementoblasts and root resorption.

Force distribution is more important than its intensity!

A common question about root resorption is raised in orthodontic practice: What is more important, the intensity of force or its distribution along the root, periodontal and alveolar structures? Diffuse distribution of forces applied to periodontal tissues during tooth movement tends not to promote neither extensive areas of cell matrix hyalinization nor significant death of cementoblasts that lead to root resorption. However, focal distribution or concentration of forces within a restricted area - as it occurs in tipping movements, even with forces of lower intensity - tend to induce extensive areas of hyalinization and focal death of cementoblasts, which is commonly associated with root resorption. In tipping movements, the apical regions tend to concentrate more forces in addition to wounding the cementoblasts due to the smaller dimension of their root structure as well as their cone shape. For this reason, there is an increase in root resorption. In the cervical region, on the other hand, the large area resulting from a large diameter and bone crown deflection tends to reduce the effects of forces, even when they are more concentrated, thus rarely inducing death of cementoblasts and root resorption.

Um questionamento comum sobre as reabsorções radiculares na prática ortodôntica: "O que é mais importante? A intensidade das forças aplicadas ou sua distribuição ao longo das estruturas radiculares, periodontais e alveolares?" A distribuição difusa das forças aplicadas sobre os tecidos periodontais durante o movimento dentário de corpo tende a não promover extensas áreas de hialinização da matriz extracelular, nem morte significativa de cementoblastos que levariam à reabsorção radicular. Porém, a distribuição focal ou concentração de forças - como nas inclinações, mesmo nas de menor intensidade - em uma área restrita tende a induzir áreas extensas de hialinização e morte focal de cementoblastos, associando-se mais comumente à reabsorção radicular. Nos movimentos de inclinação, as áreas apicais, por sua menor dimensão da estrutura radicular e sua forma cônica, tendem a concentrar mais ainda as forças e lesar cementoblastos, aumentando a frequência das reabsorções radiculares. Na região cervical, a maior área decorrente do maior diâmetro e a deflexão óssea da crista óssea tendem a reduzir os efeitos das forças, mesmo quando mais concentradas, muito raramente induzindo a morte de cementoblastos e reabsorções radiculares.

Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities.

Compressive force induces VEGF production in periodontal tissues.

During orthodontic tooth movement, the activation of the vascular system in the compressed periodontal ligament (PDL) is an indispensable process in tissue remodeling. We hypothesized that compressive force would induce angiogenesis of PDL through the production of vascular endothelial growth factor (VEGF). We examined the localization of VEGF in rat periodontal tissues during experimental tooth movement in vivo, and the effects of continuous compressive force on VEGF production and angiogenic activity in human PDL cells in vitro. PDL cells adjacent to hyalinized tissue and alveolar bone on the compressive side showed marked VEGF immunoreactivity. VEGF mRNA expression and production in PDL cells increased, and conditioned medium stimulated tube formation. These results indicate that continuous compressive force enhances VEGF production and angiogenic activity in PDL cells, which may contribute to periodontal remodeling, including angiogenesis, during orthodontic tooth movement.

Orthodontic mechanotransduction and the role of the P2X7 receptor.

INTRODUCTION: The P2X7 receptor plays a crucial role in bone biology and inflammation. Its main function is to promote necrotic tissue metabolism by ensuring a normal acute-phase inflammatory response. We used a mouse model to describe and compare orthodontic mechanotransduction in wild-type and P2X7 knock-out mice. METHODS: By using finite element analysis, mouse orthodontic mechanics were scaled to produce typical human stress levels. External root resorption, bone modeling, and bone remodeling were analyzed with fluorescent bone labels, Masson trichrome stain, and microcomputed tomography. Relationships between the biologic responses and the calculated stresses were statistically tested and compared between mouse types. RESULTS: There were direct relationships between certain stress magnitudes and root resorption and bone formation. Hyalinization and root and bone resorption were different in the 2 types of mice. CONCLUSIONS: Orthodontic responses are related to the principal stress patterns in the periodontal ligament, and the P2X7 receptor plays a significant role in their mechanotransduction.

Histopathology of nonotosclerotic stapes fixations.

HYPOTHESIS: Different diseases without exact histopathologic classification can cause stapes ankylosis. BACKGROUND: Otosclerosis is a complex bone remodeling disorder of the otic capsule due to persisting measles virus infection and consecutive inflammatory reaction. In fact, clinical and demographic features of otosclerosis have reference to stapes ankylosis. In the clinical practice, otosclerosis and stapes ankylosis are incorrect synonyms. METHODS: Nonotosclerotic stapes footplates (n = 284) removed during stapedectomy were analyzed histologically. Otosclerosis was excluded during the histologic preselection (n = 437). Total RNA was extracted, and measles virus-specific reverse-transcriptase-polymerase chain reaction was performed. RESULTS: Nonotosclerotic stapes ankylosis was associated with total absence of measles virus RNA. Six main types of nonotosclerotic stapes fixations could be distinguished histologically: annular calcification (n = 152; 53.5%), globular fibrosis (n = 49; 17.25%), lymphocytic infiltration (n = 31; 10.9%), hemosiderosis (n = 22; 7.75%), granulomas (n = 17; 6%) and amyloidosis (n = 13; 4.6%). Fragmentation of nonotosclerotic stapes footplates was infrequent (7%) during stapes surgery. Only 1 floating footplate (0.35%) was reported. CONCLUSION: Two thirds of nonotosclerotic stapes footplates represented complete pathologic bone remodeling. Unlike otosclerosis, nonotosclerotic stapes fixations were characterized by basic histopathologic findings without organ specificity that can also be identified in case of different diseases. Prevalence of nonotosclerotic stapes ankylosis is approximately 30 to 40% among stapes fixation cases. The long-term prognosis and surgical considerations theoretically differ from those of otosclerosis.

Hyalinization during orthodontic tooth movement: a systematic review on tissue reactions.

The purpose of this study was to perform a meta-analysis on the literature concerning hyalinization in relation to experimental tooth movement in animals and humans. A structured search of electronic databases as well as hand searching retrieved 70 publications concerning the subject. After application of inclusion and exclusion criteria, 39 studies remained, of which three were in humans. Articles on animal experiments were in the majority with most studies performed in rats. Among other data force magnitude, type of tooth movement, duration of the experimental period, and moment of first and last appearance of hyalinization were extracted from the included studies. The heterogeneity of the published studies and the limited data on appearance of hyalinization made it impossible to perform a meta-analysis. Therefore, the literature was systematically reviewed. It appears that there are no major differences in tissue reaction between species during experimental tooth movement. Although hyalinization is considered to be an undesirable side-effect of orthodontic tooth movement, little attention has been paid to the phenomenon itself and its possible relationship with stress/strain levels in the periodontal ligament (PDL) and alveolar bone or the rate after the initial phase of tooth movement. There is a need for well-designed experimental studies to elucidate the role of hyalinization in orthodontic tooth movement.

Basic science of articular cartilage and osteoarthritis.

Articular cartilage is a specialized tissue uniquely suited for load distribution with a low-friction articulating surface. Its compressive and tensile properties are determined by its matrix and fluid composition, and are maintained by chondrocytes in the homeostatic joint. Osteoarthritis (OA) is increasingly understood as a family of disorders in which the biomechanical properties of cartilage are altered and ultimately fail as the tissue is degraded by local proteases. Mechanically mediated and cytokine-mediated pathways of cartilage degeneration have been identified in the pathogenesis of OA. Further insight into the basic science of cartilage and OA is necessary to develop diagnostic and treatment strategies for this pervasive disease.

Triple-phase dynamic MRI of intratumoral vessel density and hyalinization grade in uterine leiomyomas.

OBJECTIVE: Our purpose was to clarify the relationship between the tissue vascularity shown on triple-phase dynamic MRI and the number of intratumoral vessels and degree of hyalinization, which are two histopathologic changes in leiomyoma. SUBJECTS AND METHODS. The subjects were 10 premenopausal patients with 20 leiomyomas who had undergone surgery without preoperative gonadotropin-releasing hormone analogue treatment. Intratumoral vessel density was determined by the mean number of intratumoral vessels with at least one smooth-muscle layer in the optic fields magnified 100 times. Hyalinization grade was determined by the severity of hyalinization, histopathologically classified in three grades. The enhancement index (EI) of the leiomyoma was calculated using the formula EI(t) = [S(t) - S(0)] / S(0), where S(0) is the signal intensity on pre-enhanced T1-weighted images and S(t) is the signal intensity on each dynamic phase image (t = 20, 60, and 180 sec) [corrected]. The histopathologic parameters of intratumoral vessel density and hyalinization grade were compared with the enhancement indexes obtained from the triple-phase dynamic MRI. RESULTS: We found positive correlations between intratumoral vessel density and EI(60) and between intratumoral vessel density and EI(180) (in both cases, p = 0.0028 and r = 0.69). We found significant differences among the mean enhancement indexes for each hyalinization grade at all dynamic phases (p < 0.01). The leiomyomas with lower intratumoral vessel densities tended to show greater hyalinization. CONCLUSION: Our results showed that leiomyomas with only slight hyalinization or with abundant vessels were well enhanced, but the leiomyomas with severe hyalinization enhanced poorly.

Design characteristics for the tissue engineering of cartilaginous tissues.

Tissues like the temporomandibular joint (TMJ) disc and the knee meniscus are often mistakenly viewed as a tantamount to hyaline cartilage, largely due to the absence of a comprehensive understanding of the distinguishing properties of cartilaginous tissues. Because of this confusion, fibrocartilaginous tissue engineering attempts may not be based on suitable experimental designs. Fibrocartilaginous tissues are markedly different than hyaline cartilage; however, the dearth of knowledge related to their cellular and biochemical composition, as well as their biomechanical characteristics, is stunning. Hyaline articular cartilage is exclusively composed of chondrocytes that produce primarily type II collagen, whereas the TMJ disc and the knee meniscus have a mixed cell population of fibroblasts and cells similar to chondrocytes, which predominantly secrete type I collagen. Additionally, fibrocartilaginous tissues have a low glycosaminoglycan content, a low compressive modulus, and a high tensile modulus when compared to hyaline cartilage. Therefore, it is crucial for fibrocartilaginous tissue engineering attempts to be tissue-specific, utilizing the knowledge of the distinct and unique properties of these tissues. At the same time, advances and insights related to the science and engineering aspect of hyaline cartilage regeneration must be carefully considered for the in vitro engineering of fibrocartilaginous tissues.

Magnetic resonance imaging of hyaline cartilage regeneration in neocartilage graft implantation.

BACKGROUND: The purpose of this study was to investigate the regenerative potential of hyaline cartilage in a neocartilage graft implant with the aid of MR cartilage imaging using a rabbit model. METHODS: Surgical osteochondral defects were created in the femoral condyles of 30 mature New Zealand rabbits. The findings of neocartilage in autologous cartilage grafts packed into osteochondral defects were compared with control group of no implant to the osteochondral defect. The outcome of the implantations was correlated with histologic and MR cartilage imaging findings over a 3-month interval. RESULTS: Neocartilage grafts packed into osteochondral defects showed regeneration of hyaline cartilage at the outer layer of the implant using MR cartilage imaging. Fibrosis of fibrocartilage developed at the outer layer of the autologous cartilage graft together with an inflammatory reaction within the osteochondral defect. CONCLUSION: This animal study provides evidence of the regenerative ability of hyaline cartilage in neocartilage transplants to repair articular cartilage.

Subchondral bone and cartilage disease: a rediscovered functional unit.

The role of subchondral bone in the pathogenesis of cartilage damage has likely been underestimated. Subchondral bone is not only an important shock absorber, but it may also be important for cartilage metabolism. Contrary to many drawings and published reports, the subchondral region is highly vascularized and vulnerable. Its terminal vessels have, in part, direct contact with the deepest hyaline cartilage layer. The perfusion of these vessels accounts for more than 50% of the glucose, oxygen, and water requirements of cartilage. Bony structure, local metabolism, hemodynamics, and vascularization of the subchondral region differ within a single joint and from one joint to another. Owing to these differences, repetitive, chronic overloading or perfusion abnormalities may result in no pathological reaction at all in one joint, while in another joint, these same conditions may lead to osteonecrosis, osteochondritis dissecans, or degenerative changes. According to this common etiological root, similar pathological reactions beginning with marrow edema and necrosis and followed by bone and cartilage fractures, joint deformity, and insufficient healing processes are found in osteonecrosis, osteochondritis dissecans, and degenerative disease as well.

Plasminogen did not affect lung function in surfactant-treated preterm lambs.

Preterm infants with respiratory distress syndrome develop fibrin-rich hyaline membranes within the alveoli and have depressed fibrinolytic activity, which is thought to be due to a relative deficiency of plasminogen. Local fibrin deposition inhibits surfactant function and amplifies inflammation. We hypothesized that plasminogen administration to surfactant-treated preterm lambs would prevent fibrin-rich hyaline membrane formation, resulting in the amelioration of lung pathology and improved lung function. We randomly treated preterm lambs (gestational age 127-129 days) with either 16 mg of lysine-plasminogen (n = 10) or saline (n = 10), and ventilated them for 5 h. There were no significant differences in physiologic measurements of lung function (ventilation efficiency index, oxygenation index, dynamic compliance, quasi-static pressure volume curve), measures of lung injury (alveolar wash protein content and (125)I-albumin recovery) or surfactant pool size. The degree and extent of bronchiolar erosion and hyaline membrane formation were similar in the two groups. Plasminogen administration did not improve lung function or prevent hyaline membrane formation in surfactant-treated lambs.