Specific inductive potential of a novel nanocomposite biomimetic biomaterial for osteochondral tissue regeneration.

Osteochondral lesions require treatment to restore the biology and functionality of the joint. A novel nanostructured biomimetic gradient scaffold was developed to mimic the biochemical and biophysical properties of the different layers of native osteochondral structure. The present results show that the scaffold presents important physicochemical characteristics and can support the growth and differentiation of mesenchymal stromal cells (h-MSCs), which adhere and penetrate into the cartilaginous and bony layers. H-MSCs grown in chondrogenic or osteogenic medium decreased their proliferation during days 14-52 on both scaffold layers and in medium without inducing factors used as controls. Both chondrogenic and osteogenic differentiation of h-MSCs occurred from day 28 and were increased on day 52, but not in the control medium. Safranin O staining and collagen type II and proteoglycans immunostaining confirmed that chondrogenic differentiation was specifically induced only in the cartilaginous layer. Conversely, von Kossa staining, osteocalcin and osteopontin immunostaining confirmed that osteogenic differentiation occurred on both layers. This study shows the specific potential of each layer of the biomimetic scaffold to induce chondrogenic or osteogenic differentiation of h-MSCs. These processes depended mainly on the media used but not the biomaterial itself, suggesting that the local milieu is fundamental for guiding cell differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Cell death in human articular chondrocyte: a morpho-functional study in micromass model.

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.

Use of irradiate animal pericardium membrane for prevention of Frey's syndrome after parotidectomy.

BACKGROUND: Frey syndrome is a common complication that appears few months after parotid surgery with flushing and sweating of the parotid-temporal area during mastication. It presumably originates from an aberrant nervous regeneration in which the parasympathetic fibers of the parotid gland would combine themselves with the sympathetic fibers of the sweat glands and with the cutaneous vessels. AIM: In the present study we analyze the effectiveness of a collagenous membrane derived from animal pericardium (APM) to prevent Frey's syndrome after parotidectomy. MATERIALS AND METHODS: We studied a total of 40 patients with benign tumors of the parotid gland, including 30 patients with pleomorphic adenoma, 7 patients with Warthin tumor and 3 with basal cells adenoma. The patients were divided into 2 groups: group 1 (experimental n=20) executed superficial parotidectomy with replacement of bovine pericardial matrix (BPM); group 2 (control n=20) underwent superficial parotidectomy followed by reposition of superficial musculoaponeurotic system (SMAS) flap. All patients were questioned over their subjective symptom and tested with Minor's test after 12 months from the intervention and introduced in a follow-up of 3 years. RESULTS: Subjectively Frey syndrome was referred in 5% of patients in group 1 and in 10% in group 2, while 0 cases were observed in group 1 after the starch-iodine test, 2 cases in group 2 (10%). CONCLUSIONS: Considering the present results, although this study needs further implementation, we can affirm that BPM is a valid option in preventing Frey's syndrome whereas SMAS flap is not available.

Bone marrow concentrated cell transplantation: rationale for its use in the treatment of human osteochondral lesions.

Bone marrow is one of the best characterized stem cell microenvironments that contains Mesenchymal Stem Cells (MSCs), a rare population of non-hematopoietic stromal cells. MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages such as bone, cartilage and adipose tissue. However, isolation procedures are crucial for the functional activity of the transplanted cells. The use of concentrated bone marrow cells (BMCs) enables a cell population surrounded by its microenvironment (niche) to be implanted while avoiding all the complications related to the in vitro culture. The cells of the niche are able to regulate stem cell behavior through direct physical contact and secreting paracrine factors. The aim of this study was to characterize BMCs in vitro to evaluate their ability to differentiate toward mature cells and try to understand whether there are differences in the chondrogenic and osteogenic potential of cells from patients of different ages. Mononuclear Cells (MNCs) isolated by Ficoll were used as control. Both cell populations were grown in monolayers and differentiated with specific factors and analyzed by histological and molecular biology assays to evaluate the expression of some specific extracellular matrix molecules. The present investigations revealed the ability of BMCs to act as isolated cells. They are able to form colonies and differentiate toward chondrogenic and osteogenic lineages, the latter pathway appearing to be influenced by donor age. The results obtained by this study support the use of BMCs in clinical practice for the repair of osteochondral damage, which might be particularly useful for the one-step procedure allowing cells to be directly implanted in operating room.

Treatment of human cartilage defects by means of Nd:YAG Laser Therapy.

Articular cartilage lesions represent a challenging problem for orthopaedic surgeons. The purpose of this study was to evaluate the effect of a new pulsed Nd:YAG High Intensity Laser Therapy on the regeneration of cartilage tissue in patients with traumatic lesions. Clinical, histological and immunohistochemical evaluations were performed. Ten patients affected by chondral lesions scheduled for ACI procedure, were enrolled into the study. During the chondrocyte expansion for ACI procedure, cartilage from five patients was treated by Nd:YAG High Intensity Laser Therapy (HILT group). No laser treatment was performed in the remaining patients, who were used as controls. Cartilage repair was assessed by clinicians using two different scores: Cartilage Repair Assessment (CRA) and Overall Repair Assessment (ORA). Cartilage biopsy specimens were harvested to perform histological and immunohistochemical analyses at T0 (before laser treatment) and T1 (at the end of the treatment). A significant decrease in cartilage depth was noticed in the HILT group at T1. Histological and immunohistochemical evaluations showed some regenerative processes in cartilaginous tissue in terms of high amount of proteoglycans, integration with adjacent articular cartilage and good cellular arrangement in the HILT group. By contrast, a not well organized cartilaginous tissue with various fibrous features in the control group at T0 and T1 was observed. In conclusion, the use of this new pulsed Nd:YAG HILT resulted promising in the treatment of moderate cartilage lesions markedly in the young patients.

An orbital roof and anterior skull base fracture: case report.

Blow-out fractures usually involve the orbit in the floor or in the medial wall. Anyway, if the roof of the orbit is thin and direct compressive or buckling forces impact the orbit the fracture can involve the upper roof. We describe the case of a blow-out fracture of the orbital roof with enophtalmus and cerebrospinal fluid leak from lacero-contusive subciliar wound

Osteonecrosis of the jaws in patients assuming bisphosphonates and sunitinib: two case reports.

Osteonecrosis of the jaw (ONJ) is an unremitting adverse outcome associated with bisphosphonate therapy, primarily intravenously administered, in patients with bone metastases from solid tumors, multiple myeloma and osteometabolic diseases. From 2003 many cases of bisphosphonates related osteonecrosis of the jaw (BRONJ) have been reported in literature. Sunititnib is a novel anticancer agent used in gastrointestinal cancers and renal cancers resistant to imatinib. Recent reports describe the onset of ONJ in patients treated with both sunitinib and bisphosponates. A case of osteonecrosis of the jaw related to sunitinib, without association of bisphosphonate (BP) medications has been recently reported. A recent hypothesis suggests that antiangiogenic drugs such as sunitinib could cause ONJ even without the association with BPs. We describe a case of two patients affected by renal carcinoma under BP and sunitinib medication who developed stage III bisphosphonates-related osteonecrosis of the jaw (BRONJ).

Induced sputum in the management of COPD: clinical implications.

Sputum induction can be used as a non-invasive technique to investigate airway inflammation in asthma and COPD. We reported the case of a 68 year old man with COPD, stage III GOLD, that underwent sputum induction during two exacerbation episodes. The first cell count showed a typical sputum neutrophilia, whereas the second showed sputum eosinophilia. On the basis of sputum cellularity, we decided to treat the first episode with a course of antibiotics and the second exacerbation with a course of antibiotics and oral steroids. The patient showed improvement in both cases, obtaining clinical stabilisation. The induced sputum cell count could be a useful technique in a clinical setting to evaluate the cellular characteristics of airway inflammation during COPD exacerbation and modulate the antinflammatory therapy.

Clinical applications and biosafety of human adult mesenchymal stem cells.

Mesenchymal Stem Cells (MSCs) are a population of adherent cells that can differentiate into mesenchymal lineage populations (cartilage, bone and fat tissue). In addition, they seem to be able to differentiate also into a broader type of lineages other than the original mesodermal germ layer. Bone marrow MSCs are a standard in the field of adult stem cell biology and clinical applications; however adipose-derived MSCs are becoming an attractive alternative due to their minimally invasive accessibility and availability in the body. MSCs modulate several effector immune functions by interacting both with innate and adoptive immune responses. Several local signals from the tissue microenvironment, together with cytokine and soluble factors released by MSCs influence anti-inflammatory and tissue repair properties of infused MSCs. Therefore, cellular therapies utilizing ex vivo expanded MSCs may be an interesting approach for inflammatory and autoimmune diseases. Biosafety is still one of the most important aspects; therefore the production of clinical-grade MSCs requires the careful identification and control of all the phases of cell manipulation and release. Many clinical applications of adult MSCs are in progress and are using bone marrow or adipose tissue-derived MSCs for the treatment of Graft Versus Host Disease (GVHD), inflammatory joint diseases and osteocartilagineous defects, digestive tract, cardiovascular and neurological diseases.

Nuclear damages and oxidative stress: new perspectives for laminopathies.

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

Role of polyamines in hypertrophy and terminal differentiation of osteoarthritic chondrocytes.

Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Chemical-physical properties and in vitro cell culturing of a novel biphasic bio-mimetic scaffold for osteo-chondral tissue regeneration.

The requirements for a successful regeneration of an osteo-chondral defect could effectively be met by using a bi-layered composite scaffold, able to support proliferation and differentiation of mesenchymal stem cells, while providing a biochemical environment promoting the formations of the two distinct tissues. The novel strategy here presented consists of developing a bio-mimetic scaffolds obtained by the combination of two integrated organic compounds (type I collagen and chitosan) with or without bioactive Mg-doped hydroxyapatite (Mg-HA) nanocrystals, depending on the specific layer, reproducing cartilaginous or subchondral bone tissue. An innovative patented methodology for scaffolds production, called - pH-dependent 3-phasic assembling -, allowed to development of a highly homogenous and chemically stable scaffold, presenting a very good integration among all three components, as confirmed by extensive SEM and thermogravimetric analyses. A preliminary in vitro evaluation was also carried out by seeding bi-layered scaffold with human bone marrow stromal cells (h-MSCs), by giving particular emphasis to cell viability and distribution at day 0, 7 and 14. Cells were viable and uniformly colonized the whole scaffold until day 14, indicating that the scaffold contributed to the maintenance of cell behaviour.

Evaluation of chondrocyte behavior in a new equine collagen scaffold useful for cartilage repair.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

Interleukin-4/interleukin-4 receptor gene polymorphisms in hand osteoarthritis.

OBJECTIVE: IL-13/IL-4/IL-4R system has strong chondroprotective activity. We investigated polymorphisms in these genes as potential hand osteoarthritis (OA) susceptibility loci by performing a case-control association study. METHODS: Eighteen common single nucleotide polymorphisms (SNPs) (nine in IL-4R, five in IL-4 and four in IL-13) were genotyped in 403 patients (380 females) with hand OA and 322 healthy controls (308 females). RESULTS: Two SNPs (rs1805013 and rs1805015), mapping to the IL-4R gene, were associated with P-values of 0.0116 and 0.0305 respectively in the whole sample. As far as the non-erosive hand OA group (n=159) is concerned, the significance level of association of SNP rs1805013 is increased. After correction for multiple testing (correction for the 54 tests) the significance was not retained. None of the IL-13 SNPs analyzed showed association with hand OA. Some of the analyzed SNP within the IL-4 gene showed significant association with hand OA only when considering subgroups of patients. With respect to the CMC1 OA group, two SNPs in IL-4 (rs2243250 and rs2243274) showed association with a P-value of 0.027 and 0.018 respectively. None of these associations remained after correction for multiple testing. CONCLUSIONS: The present study shows a trend to an association between non-erosive hand OA in Caucasian population and a genetic variant in the coding region of IL-4R gene. Our results, in keeping with previous data on hip OA, confirm the suggestion that IL-4/IL-4R system plays a role in OA pathogenesis. Further confirmation studies on different populations are necessary.

Difluoromethylornithine inhibits hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone in cardiac cells.

Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (D, L)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.

Ligament repair: a molecular and immunohistological characterization.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during "ligamentization". Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction.

Circulating RANKL/OPG in polymyalgia rheumatica.

OBJECTIVE: To evaluate whether RANKL/OPG balance is modified in PMR patients, either in the active phase of the disease or during corticosteroid treatment. METHODS: Circulating levels of RANKL and OPG were assayed by enzyme-linked immunosorbent assay in PMR patients with active untreated disease and in patients treated by corticosteroids over a 12-month follow-up period. RESULTS: We found no statistically significant differences in circulating levels of OPG between PMR patients either in the active phase of the disease or during all follow-up period compared to normal controls. On the other hand, systemic production of sRANKL is increased and is not modulated by corticosteroid treatment. CONCLUSION: In PMR increased levels of sRANKL may be related to bone osteoporosis. Further investigations are necessary to evaluate the relationship between the RANK/RANKL/OPG system and bone turnover in PMR patients.

A molecular and histological characterization of cartilage from patients with Morquio syndrome.

OBJECTIVE: To investigate the gene expression profile and the histological aspects of articular cartilage of patients affected by Morquio syndrome, a lysosomal storage disease characterized by the accumulation of glycosaminoglycans within the cells which result in abnormal formation and growth of the skeletal system. METHOD: Articular cartilage samples were obtained from the femoral condyle of two siblings with Morquio syndrome during surgery performed to treat valgus knee. As controls, four biopsy samples of healthy cartilage were obtained from four different male multiorgan donors. A Real-Time Polymerase Chain reaction (RT-PCR) analysis was performed to evaluate the expression of type I and II collagens and aggrecan mRNAs. Histological and immunohistochemical analyses for some matrix proteins were carried out on paraffin embedded sections. RESULTS: Type I collagen mRNA mean level was higher in the samples of patients with Morquio syndrome compared to controls. Type II collagen and aggrecan mRNAs' mean expression was instead lower. The morphological appearance of the cartilage showed a poorly organized tissue structure with not homogeneously distributed cells that were larger compared to normal chondrocytes due to the presence inside the vacuoles of proteoglycans which were not metabolized. Chondrocytes were negative for collagen II immunostaining while the extracellular matrix was weakly positive. Collagen type I immunostaining was positive at cellular level. Keratan sulfate showed diffuse positivity and chondroitin-6-sulfate was present throughout the cartilaginous thickness. CONCLUSION: In cartilage of patients with Morquio syndrome, a low expression of collagen type II and a high expression of collagen type I both at protein and molecular levels are evidentiated. This finding could give evidence of the reduction in ankle and knee joint movement observable in these patients.

Polyamine biosynthesis as a target to inhibit apoptosis of non-tumoral cells.

Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. alpha-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.