C. Loreto

New perspectives for articular cartilage repair treatment through tissue engineering: A contemporary review

In this paper review we describe benefits and disadvantages of the established methods of cartilage regeneration that seem to have a better long-term effectiveness. We illustrated the anatomical aspect of the knee joint cartilage, the current state of cartilage tissue engineering, through mesenchymal stem cells and biomaterials, and in conclusion we provide a short overview on the rehabilitation after articular cartilage repair procedures. Adult articular cartilage has low capacity to repair itself, and thus even minor injuries may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. Numerous efforts have been made to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, and to date several researchers aim to implement clinical application of cell-based therapies for cartilage repair. A literature review was conducted on PubMed, Scopus and Google Scholar using appropriate keywords, examining the current literature on the well-known tissue engineering methods for the treatment of knee osteoarthritis.

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.

Physical activity ameliorates cartilage degeneration in a rat model of aging: A study on lubricin expression

Osteoarthritis (OA) is a common musculoskeletal disorder characterized by slow progression and joint tissue degeneration. Aging is one of the most prominent risk factors for the development and progression of OA. OA is not, however, an inevitable consequence of aging and age‐related changes in the joint can be distinguished from those that are the result of joint injury or inflammatory disease. The question that remains is whether OA can be prevented by undertaking regular physical activity. Would moderate physical activity in the elderly cartilage (and lubricin expression) comparable to a sedentary healthy adult? In this study we used physical exercise in healthy young, adult, and aged rats to evaluate the expression of lubricin as a novel biomarker of chondrocyte senescence. Immunohistochemistry and western blotting were used to evaluate the expression of lubricin in articular cartilage, while enzyme‐linked immunosorbent assay was used to quantify lubricin in synovial fluid. Morphological evaluation was done by histology to monitor possible tissue alterations. Our data suggest that moderate physical activity and normal mechanical joint loading in elderly rats improve tribology and lubricative properties of articular cartilage, promoting lubricin synthesis and its elevation in synovial fluid, thus preventing cartilage degradation compared with unexercised adult rats.

Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: An in vivo and in vitro study

Osteoarthritis (OA) of the knee is a major problem in our society. The development of new treatment options for OA is limited, because the pathophysiological mechanisms are not clearly understood, especially on the molecular level. Aquaporin 1 (AQP1) is a specific protein channels for water transport; it is expressed in articular chondrocytes, human synovitis, in chondrocytes of patients with rheumatoid arthritis or OA and in chondrocyte-like cells of human intervertebral disc. The aim of this study was to investigate the expression of AQP1, through immunohistochemistry, immunocytochemistry and Western blot, in experimentally induced OA knee menisci. AQP1 was studied in vivo in knee OA menisci from 36 rats that underwent medial or lateral meniscectomy, and in vitro on fibrochondrocytes derived from knee OA menisci rats. OA in rats was experimentally induced and tested by histomorphometric analysis. Histological results demonstrated structural alterations in OA menisci accompanied by a very strong AQP1 immunohistochemical and immunocytochemical staining. The Western blot analysis confirmed a strong expression of AQP1 in OA fibrochondrocytes cells. The results of the present research suggest that an activation of AQP1, induced by the OA process, may represent an endogenous mechanism, which can be used to control the tissue degeneration within OA articular joints.

An ex vivo study on immunohistochemical localization of MMP-7 and MMP-9 in temporomandibular joint discs with internal derangement

Internal derangement (ID) is among the most common disorders of the temporomandibular joint (TMJ). Previous research by our group highlighted a correlation between apoptosis and TMJ ID. Metalloproteinases (MMP)-7 and -9 have been shown to play an important role in extracellular matrix ECM) homeostasis and, through it, in joint disc remodelling. The immunohistochemical expression of MMP-7 and -9 was investigated in discs from patients with TMJ ID and from healthy donors and compared with the degree of histological tissue degeneration. The collagen fibre arrangement in pathological discs exhibited varying degrees of disruption. New vessels were consistently detected; endothelial cells from these vessels were immunolabelled with both MMP-7 and MMP-9. More or less intense MMP-7 and MMP-9 immunolabelling was detected in the cytoplasm of disc cells from all patients. MMP-7 and MMP-9 immunostaining was significantly different between pathological and normal discs and correlated with the extent of histopathological degeneration. MMP-7 and MMP-9 upregulation in discs from patients with TMJ ID demonstrates their involvement in disc damage in this disorder. A greater understanding of these processes could help identify ways to curb MMP overproduction without affecting their tissue remodelling action. The design of specific inhibitors for these MMPs would not only help to gain insights into the biological roles of MMPs, but would also aid in developing therapeutic interventions for diseases associated with abnormal ECM degradation.

β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker.

Degenerative disc disease of herniated intervertebral discs is associated with extracellular matrix remodeling, vimentin-positive cells and cell death

We studied patients with degenerative disc disease (DDD) to demonstrate that i) remodeling of the extracellular matrix (ECM) in the intervertebral disc (IVD), particularly the elastic fiber system, of subjects with herniated discs is dysregulated and that ii) it is accompanied by accelerated elastin degradation due to increased expression of matrix metalloprotease-9 (MMP-9). Moreover we wanted to obtain a deeper insight into the pathogenesis of DDD through the study of ECM calcification, DNA fragmentation using TUNEL analysis, BAX, bcl-2 and vimentin immunopositive cells. We studied herniated discs from patients of three age groups (group 1=30-40 years; group 2=40-50 years; and group 3=50-65 years) to evaluate the oxytalan fiber systemMMP-9, apoptosis and vimentin immunopositive cells. The results demonstrated the presence of oxytalan fibers in the annulus fibrosus (AF) and the nucleus pulposus (NP) of herniated discs. In the AF oxytalan fibers replaced disrupted mature elastic fibers in calcified areas, while in the NP they were mostly found in nests at the periphery of chondrocytes. MMP-9 was prevalently observed in NP nests above all in group 1 and group 3 discs while group 2 exhibited a lower MMP-9 immunostaining. Activation of the apoptotic process was demonstrated by upregulated BAX expression in group 3. BAX immunopositivity was inversely mirrored by a significant decrease in bcl-2 expression. Intermediate filament protein vimentin was strongly expressed only in group 1 samples. A large number of apoptotic TUNEL+ cells was observed in group 3 specimens. The presence of oxytalan fibers may be the result of a process of incomplete elastogenesis, or a response to mechanical stress trying to functionally replace the lack of elastic fibers. MMP-9 expression seems to relate to disc damage, while chondrocyte BAX upregulation and TUNEL+ cell staining revealed apoptosis activation regardless of patient age. Vimentin immunopositivity was clearly detected in group 1 annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs.

Histochemistry as a unique approach for investigating normal and osteoarthritic cartilage

In this review article, we describe benefits and disadvantages of the established histochemical methods for studying articular cartilage tissue under normal, pathological and experimental conditions. We illustrate the current knowledge on cartilage tissue based on histological and immunohistochemical aspects, and in conclusion we provide a short overview on the degeneration of cartilage, such as osteoarthritis. Adult articular cartilage has low capacity to repair itself, and thus even minor injuries may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. Numerous efforts have been made to implement the knowledge in the study of cartilage in the last years, and histochemistry proved to be an especially powerful tool to this aim.

Advantages of exercise in rehabilitation, treatment and prevention of altered morphological features in knee osteoarthritis. A narrative review.

Knee osteoarthritis (OA) represents one of the most common causes of disability in the world. It leads to social, psychological and economic costs with financial consequences, also because a further increase is expected. Different knee OA treatments are usually considered in relation to the stage of the disease, such as surgical management and pharmacologic and non-pharmacologic treatments. Treatment should begin with the safest and least invasive one, before proceeding to more invasive, expensive ones. Non-pharmacologic, behavioral treatments of knee OA are recommended not only in rehabilitation but also in prevention because many risk factors, such as excess weight, obesity and joint tissue inflammation, can be monitored and thus prevented. In the present review, we analyze data from the most recent literature in relation to the effects of physical exercise on prevention, therapy and rehabilitation in knee OA. All data suggest that physical exercise is an effective, economical and accessible tool to everyone, in the treatment and prevention of knee OA. The literature search was conducted on PubMed, Scopus and Google Scholar using appropriate keywords in relation to knee osteoarthritis.

Immunohistochemical analysis of matrix metalloproteinase-13 in human caries dentin

The immunoexpression profile of matrix metalloproteinase-13 was investigated for the first time in dentin of human caries and healthy teeth. Twelve permanent premolars (10 caries and 2 sound) were decalcified in ethylenediaminetetraacetic acid and processed for embedding in paraffin wax. Sections 3-4 µm in thickness were cut and processed for immunohistochemistry. A mouse monoclonal anti-metalloproteinase-13 antibody was used for localisation using an immunoperoxidase technique. Dentinal immunoreactivity was detected in all teeth; it was weak in sound teeth and strong close to the caries area. These in vivo findings suggest a role for metalloproteinase-13 in the development and progression of adult human dental tissue disorders.