Concentrated adipose tissue infusion for the treatment of knee osteoarthritis: clinical and histological observations.

PURPOSE: Osteoarthritis (OA) is characterized by articular cartilage degeneration and subchondral bone sclerosis. OA can benefit of non-surgical treatments with collagenase-isolated stromal vascular fraction (SVF) or cultured-expanded mesenchymal stem cells (ASCs). To avoid high manipulation of the lipoaspirate needed to obtain ASCs and SVF, we investigated whether articular infusions of autologous concentrated adipose tissue are an effective treatment for knee OA patients. METHODS: The knee of 20 OA patients was intra-articularly injected with autologous concentrated adipose tissue, obtained after centrifugation of lipoaspirate. Patients' articular functionality and pain were evaluated by VAS and WOMAC scores at three, six and 18 months from infusion. The osteogenic and chondrogenic ability of ASCs contained in the injected adipose tissue was studied in in vitro primary osteoblast and chondrocyte cell cultures, also plated on 3D-bone scaffold. Knee articular biopsies of patients previously treated with adipose tissue were analyzed. Immunohistochemistry (IHC) and scanning electron microscopy (SEM) were performed to detect cell differentiation and tissue regeneration. RESULTS: The treatment resulted safe, and all patients reported an improvement in terms of pain reduction and increase of function. According to the osteogenic or chondrogenic stimulation, ASCs expressed alkaline phosphatase or aggrecan, respectively. The presence of a layer of newly formed tissue was visualized by IHC staining and SEM. The biopsy of previously treated knee joints showed new tissue formation, starting from the bone side of the osteochondral lesion. CONCLUSIONS: Overall our data indicate that adipose tissue infusion stimulates tissue regeneration and might be considered a safe treatment for knee OA.

Absolute quantification of residual DNA in a new extracellular matrix derived from human reticular dermis (HADM) using real-time TaqMan® MGB-PCR.

BACKGROUND: The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis. OBJECTIVE: The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA. METHODS: It was compared the incubation of the dermis in NaOH (0.06 N) and in the standard culture medium "Dulbecco Modified Eagle Medium" (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA. RESULTS: It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA. CONCLUSIONS: Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.

Adipose Derived-Mesenchymal Stem Cells Viability and Differentiating Features for Orthopaedic Reparative Applications: Banking of Adipose Tissue.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at -80°C and -196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at -80°C and -196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

Regulation of Langerhans cell functions in a hypoxic environment.

UNLABELLED: Langerhans cells (LCs) are a specialized dendritic cell subset that resides in the epidermis and mucosal epithelia and is critical for the orchestration of skin immunity. Recent evidence suggest that LCs are involved in aberrant wound healing and in the development of hypertrophic scars and chronic wounds, which are characterized by a hypoxic environment. Understanding LCs biology under hypoxia may, thus, lead to the identification of novel pathogenetic mechanisms of wound repair disorders and open new therapeutic opportunities to improve wound healing. In this study, we characterize a previously unrecognized role for hypoxia in significantly affecting the phenotype and functional properties of human monocyte-derived LCs, impairing their ability to stimulate naive T cell responses, and identify the triggering receptor expressed on myeloid (TREM)-1, a member of the Ig immunoregulatory receptor family, as a new hypoxia-inducible gene in LCs and an activator of their proinflammatory and Th1-polarizing functions in a hypoxic environment. Furthermore, we provide the first evidence of TREM-1 expression in vivo in LCs infiltrating hypoxic areas of active hypertrophic scars and decubitous ulcers, pointing to a potential pathogenic role of this molecule in wound repair disorders. KEY MESSAGES: Hypoxia modulates surface molecule expression and cytokine profile in Langerhans cells. Hypoxia impairs human Langerhans cell stimulatory activity on naive T cells. Hypoxia selectively induces TREM-1 expression in human Langerhans cells. TREM-1 engagement stimulates Langerhans cell inflammatory and Th1-polarizing activity. TREM-1 is expressed in vivo in Langerhans cells infiltrating hypoxic skin lesions.

Dermis mechanical behaviour after different cell removal treatments.

Human acellular dermal matrices (HADMs) are used in reconstructive surgery as scaffolds promoting autologous tissue regeneration. Critical to the HADM ability to remodel and integrate into the host tissue is the removal of cells while maintaining an intact extracellular architecture. The objective of this work is to develop a methodology to analyse the mechanical properties of HADMs after decellularization to identify its ideal form of treatment and its duration. Two different decellularization techniques were used as a benchmark: the first is a well-established technique (incubation in NaOH for 1-7 weeks), and the second is an innovative technique developed by this research group (incubation in DMEM (Dulbecco's modified Eagle medium) for 1-7 weeks). After decellularization, the specimens underwent uniaxial tensile tests, and experimental data were represented with stress strain curves, calculating both engineering and true values. Mechanical tests have led to the identification of the optimal method (NaOH or DMEM) and duration for the decellularization treatment; differences between engineering and true values can reach 84%, but the engineering values remain useful to make comparisons, providing reliable indications with a simpler experimental set up and data processing.

Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study.

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco's Modified Eagle's Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.

Ex Vivo Dermis Mechanical Behavior in Relation to Decellularization Treatment Length.

BACKGROUND: The dermis is a commonly used source tissue for biologic scaffolds; all cellular and nuclear materials need to be removed to limit the inflammatory immune response by the host organism. The decellularization is critical because it must preserve the structural integrity of the extracellular matrix. This work has analyzed a decellularization procedure commonly followed for the dermal tissue that is a chemical treatment with sodium hydroxide. The goal of this work is to identify the optimal treatment length on the basis of structural properties. METHODS: Tensile tests have been performed on the native tissue and on tissues decellularized for 1-7 weeks in sodium hydroxide. The collected data have been analyzed through Tukey-Kramer test to assess if the mechanical properties (ultimate tensile stress and elastic modulus) of decellularized tissues were significantly different from the properties of the native tissue. These tests have been performed on specimens cut along two orthogonal directions (parallel and perpendicular to Langer's lines). RESULTS: The decellularization treatment performed with sodium hydroxide in general weakens the tissue: both the ultimate stress and the elastic modulus get lower. The structural properties along Langer lines orientation are more strongly impacted, while the structural properties orthogonal to Langer lines can be preserved with an optimal duration of the decellularization treatment that is 5-6 weeks. CONCLUSION: The duration of the decellularization treatment is critical not only to reach a complete decellularization, but also to preserve the mechanical properties of the tissue; 5-6 week treatment performed with sodium hydroxide allows preserving the mechanical properties of the native tissue perpendicularly to Langer lines orientation, and minimizing the impact of the decellularization process on the mechanical properties along the Langer lines orientation.

An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode.

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

Hypoxia up-regulates SERPINB3 through HIF-2α in human liver cancer cells.

SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

Preparation and characterization of a novel skin substitute.

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Epstein-Barr virus in cutaneous T-cell lymphomas: evaluation of the viral presence and significance in skin and peripheral blood.

The importance of viral agents in the development of cutaneous T-cell lymphomas (CTCL) is still debated. For this purpose, we retrospectively evaluated the Epstein-Barr virus (EBV) presence in Sézary syndrome (SS), mycosis fungoides (MF), inflammatory dermatoses (ID), and healthy donors (HD) using different approaches: EBV-DNA was quantified in skin biopsies and peripheral blood using real-time PCR, EBV-encoded small RNA (EBER) transcripts were detected by in situ hybridization (ISH), and latent membrane protein1-2 antigens were detected by immunohistochemistry. Skin biopsies were EBV-DNA-positive in 8/30 (27%) SS, 7/71 (10%) MF, and 2/18 (11%) ID patients and in none of the 25 normal skin samples. Positive mRNA (EBER) signals, always confined to cerebriform T lymphocytes, were found in 5/30 SS patients (17%), whereas signals in all MF and ID patients were negative. The presence of EBV-DNA in skin and blood samples was associated with a significantly lower survival in MF/SS patients. In evaluating EBV serological status, most (>70%) SS, MF, and ID patients showed a serological reactivation demonstrated by the presence of anti-EA IgG. In conclusion, although the finding of EBV-DNA in CTCL does not prove its etiopathogenetic role and may be related instead to immunosuppression, our study demonstrates that it has prognostic relevance.

Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.

Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin.

Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.

Evaluation of donor skin viability: fresh and cryopreserved skin using tetrazolioum salt assay.

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that 'take' of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60h after harvesting). 2. The same specimens (stored at 4 and 37 degrees C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12-30h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30h was, therefore, set-up. The data suggested that skin stored at 37 degrees C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 degrees C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 degrees C for 4 days. The PVI of thawed skin samples decreased dramatically within 24h, and had reached 0% within 6 days.