Matilde Tschon

Tissue engineering for total meniscal substitution : Animal study in sheep model

OBJECTIVE The aim of the study was to investigate the use of a novel hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Two different surgical implantation techniques in a sheep model were evaluated. METHODS Twenty-four skeletally mature sheep were treated with total medial meniscus replacements, while two meniscectomies served as empty controls. The animals were divided into two groups: cell-free scaffold and scaffold seeded with autologous chondrocytes. Two different surgical techniques were compared: in 12 animals, the implant was sutured to the capsule and to the meniscal ligament; in the other 12 animals, also a transtibial fixation of the horns was used. The animals were euthanized after 4 months. The specimens were assessed by gross inspection and histology. RESULTS All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. CONCLUSION The current study supports the potential of a novel HYAFF/polycaprolactone scaffold for total meniscal substitution. Seeding of the scaffolds with autologous chondrocytes provides some benefit in the extent of fibrocartilaginous tissue repair.

HIV-1 triggers apoptosis in primary osteoblasts and HOBIT cells through TNFα activation

Several HIV‐1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV‐1‐mediated direct and/or indirect effects on osteoblasts/osteoclasts cross‐talk regulation. This study investigated the effects of HIV‐1IIIb and HIV‐1ADA strains on osteoblasts using the osteoblast‐derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV‐1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV‐1 DNA and supernatant HIV‐1 RNA were not detected by real time PCR or b‐DNA assays respectively. Under the experimental conditions, even heat‐inactivated HIV‐1 or cross‐linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV‐1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up‐regulation of TNFα, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti‐TNFα polyclonal antibody tackled effectively HIV‐1 related induction of cell apoptosis. As a whole, these results indicate that HIV‐1 may impair bone mass structure homeostasis by TNFα regulated osteoblast apoptosis. J. Med. Virol. 80:1507–1514, 2008.

REGENERATIVE MEDICINE FOR THE TREATMENT OF MUSCULOSKELETAL OVERUSE INJURIES IN COMPETITION HORSES

PurposeTissue repair in musculoskeletal injuries is often a slow and sometimes incomplete process. Regenerative medicine based on the use of growth factors (GFs) and cell therapy is aimed at improving the quality and speed of tendon and ligament healing. The aim of this study was to evaluate the potential for the administration of a combination of autologous platelet-rich plasma (PRP) and freshly isolated bone marrow mononucleated cells (BMMNCs) in 13 competition horses affected by overuse musculoskeletal injuries (suspensory ligament desmopathy and superficial flexor tendinopathy) and refractory to other therapies.MethodsAfter ultrasonographic localisation of the lesion, the autologous BMMNC suspension and PRP were injected directly into the core lesion. BMMNC and platelet count as well as growth factors in PRP were measured to determine factors influencing the clinical outcome.ResultsHorses showed a marked improvement in their degree of lameness and 84.6% were able to return to competition. Among all the factors studied, the platelet concentration predicted the healing time: significantly faster recovery (p = 0.049) was observed in cases of PRP with more than 750 × 103/μl platelets.ConclusionsCompetition horses are involved in highly demanding activities, thus being a similar model for the high mechanical overload typical of human athletes. The promising results obtained suggest that this combined biological approach may be useful even for the treatment of recalcitrant overuse musculoskeletal injuries in highly demanding patients if the appropriate dose of cells and GFs is applied.

Human osteoblasts express functional CXC chemokine receptors 3 and 5: Activation by their ligands, CXCL10 and CXCL13, significantly induces alkaline phosphatase and β-N-acetylhexosaminidase release

Osteoblasts (OBs) contribute to the maintenance of bone homeostasis and their activity can be influenced by immune cells localized in bone lacunae. We investigated the expression of the chemokine receptors in isolated human OBs by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and flow cytometry, and report a novel finding, namely, that OBs express high levels of CXC chemokine receptor 3 (CXCR3) and 5 (CXCR5). Functional assays to evaluate CXCR3 and CXCR5 demonstrated that their ligands—CXCL10 and CXCL13, respectively—significantly induce the release of β‐N‐acetylhexosaminidase, an enzyme involved in endochondral ossification and bone remodeling able to degrade important extracellular matrix components. Alkaline phosphatase activity, a useful index of matrix formation was also up‐regulated by CXCL10 and CXCL13. However, OB activation by these ligands does not affect OB proliferation. Both Bordetella pertussis toxin and neutralizing anti‐CXCR3/anti‐CXCR5 monoclonal antibodies block CXCL10 and CXCL13 induction, respectively. We also demonstrated the expression of CXCL10 and CXCL13 in human bone tissue biopsies. These results indicate that both CXCR3/CXCL10 and CXCR5/CXCL13 receptor–ligand pairs may play an important role in OB activity through the specific up‐regulation of two enzymes, which are involved in the bone remodeling process. Moreover, our data suggest that OBs may play a role in the modulation of bone formation through the combined action of these two enzymes.

Adipose-derived mesenchymal stem cells for cartilage tissue engineering: State-of-The-Art in in vivo studies

Several therapeutic approaches have been developed to address hyaline cartilage regeneration, but to date, there is no universal procedure to promote the restoration of mechanical and functional properties of native cartilage, which is one of the most important challenges in orthopedic surgery. For cartilage tissue engineering, adult mesenchymal stem cells (MSCs) are considered as an alternative cell source to chondrocytes. Since little is known about adipose-derived mesenchymal stem cell (ADSC) cartilage regeneration potential, the aim of this review was to give an overview of in vivo studies about the chondrogenic potential and regeneration ability of culture-expanded ADSCs when implanted in heterotopic sites or in osteoarthritic and osteochondral defects. The review compares the different studies in terms of number of implanted cells and animals, cell harvesting sites, in vitro expansion and chondrogenic induction conditions, length of experimental time, defect dimensions, used scaffolds and post-explant analyses of the cartilage regeneration. Despite variability of the in vivo protocols, it seems that good cartilage formation and regeneration were obtained with chondrogenically predifferentiated ADSCs (1 × 10(7) cells for heterotopic cartilage formation and 1 × 10(6) cells/scaffold for cartilage defect regeneration) and polymeric scaffolds, even if many other aspects need to be clarified in future studies.

Comparative in vivo evaluation of porous and dense duplex titanium and hydroxyapatite coating with high roughnesses in different implantation environments

Ti (PG60) and Ti plus HA (HPG60) dense coatings with ultrahigh roughness (Ra: 74 +/- 8 microm and 53 +/- 18 microm, respectively) were compared to high Ti (Ti60) and Ti plus HA (HT60) high roughened porous coatings (Ra: 40 +/- 7 microm and 36 +/- 3 microm, respectively). Surfaces were implanted in cortical and trabecular bone of young adult (YOUNG), aged (AGED) and estrogen-deficient sheep (OVX) and analyzed by means of histology, histomorphometry and push-out tests 3 months after implantation. A significantly lower value in affinity index (AI) of PG60 when compared to TI60 (p < 0.01) was observed in cortical bone. In trabecular bone, lower values in AI were found in TI60 and PG60 when compared to their HA-coated surfaces (p < 0.0005). Bone ingrowth (BI) of TI60 and PG60 was significantly lower than that of the HA-coated surfaces in trabecular bone (p < 0.05). Significantly lower values in BI in OVX sheep in comparison to YOUNG sheep in both cortical and trabecular bone were observed (p < 0.05). Data showed that high roughness and Ti and HA-coated surfaces are suitable for aged and osteoporotic patients. HA coatings represent the most successful strategy in trabecular bone.

Agarose Gel as Biomaterial or Scaffold for Implantation Surgery: Characterization, Histological and Histomorphometric Study on Soft Tissue Response

Maxillofacial, orthopedic, oral, and plastic surgery require materials for tissue augmentation, guided regeneration, and tissue engineering approaches. In this study, the aim was to develop and characterize a new extrudable hydrogel, based on agarose gel (AG; 1.5% wt) and to evaluate the local effects after subcutaneous implantation in comparison with collagen and hyaluronic acid. AG chemical–physical properties were ascertained through Fourier transform infrared (FT-IR) spectroscopy and rheological analysis. In vivo subcutaneous implants were performed, and histological and histomorphometric evaluations were done at 1, 4, 12, and 16 weeks. FT-IR confirmed that spectroscopic properties were the same for the baseline agarose and rheological characterization established that AG is a weak hydrogel. Subcutaneous AG implants induced new vessels and fibrous tissue formation rich in neutrophils; the capsule thickness around AG increased until the 12th week but remained thinner than those around hyaluronic acid and collagen. At 16 weeks, the thickness of the capsule significantly decreased around all materials. This study confirmed that 1.5% wt AG possesses some of the most important features of the ideal biocompatible material: safety, effectiveness, costless, and easily obtained with specific chemical and geometrical characters; the AG can represent a finely controllable and biodegradable polymeric system for cells and drug delivery applications.

Ultrasound-Guided Injection of Platelet-Rich Plasma and Hyaluronic Acid, Separately and in Combination, for Hip Osteoarthritis: A Randomized Controlled Study.

Background: The effectiveness of intra-articular platelet-rich plasma (PRP) injections has been evaluated in knee chondroplasty and osteoarthritis (OA); however, little evidence of its efficacy in hip OA exists. Purpose: To compare the therapeutic efficacy of autologous PRP, hyaluronic acid (HA), or a combination of both (PRP+HA) in hip OA. Study Design: Randomized controlled trial; Level of evidence, 1. Methods: Patients aged between 18 and 65 years who were treated with outpatient surgery and who had hip OA and pain intensity at baseline of >20 on a 100-mm visual analog scale (VAS) were recruited for this study. Exclusion criteria were extensive surgery; presence of excessive deformities; or rheumatic, infective, cardiovascular, or immune system disorders. The primary outcome measure was a change in pain intensity as assessed by the VAS at 2, 6, and 12 months after treatment. Secondary outcome measures were the Harris Hip Score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and concentration of growth factors in PRP and their correlation with clinical outcomes. Clinical outcomes were evaluated by assessors and collectors blinded to the type of treatment administered. Results: A total of 111 patients were randomly assigned to 3 groups and received 3 weekly injections of either PRP (44 patients), PRP+HA (31 patients), or HA (36 patients). At all follow-ups, the PRP group had the lowest VAS scores. In particular, at 6-month follow-up, the mean VAS score was 21 (95% CI, 15-28) in the PRP group, 35 (95% CI, 26-45) in the PRP+HA group, and 44 (95% CI, 36-52) in the HA group (P < .0005 [PRP vs HA] and P = .007 [PRP vs PRP+HA]; F = 0.663). The WOMAC score of the PRP group was significantly better at 2-month follow-up (mean, 73; 95% CI, 68-78) and 6-month follow-up (mean, 72; 95% CI, 67-76) but not at 12-month follow-up. A significant, “moderate” correlation was found between interleukin-10 and variations of the VAS score (r = 0.392; P = .040). Significant improvements were achieved in reducing pain and ameliorating quality of life and functional recovery. Conclusion: Results indicated that intra-articular PRP injections offer a significant clinical improvement in patients with hip OA without relevant side effects. The benefit was significantly more stable up to 12 months as compared with the other tested treatments. The addition of PRP+HA did not lead to a significant improvement in pain symptoms.

Ablation of bone cells by electroporation

Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells. Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing. After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times. Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases.

In vitro and in vivo response to nanotopographically-modified surfaces of poly (3 -hydroxybutyrate -co -3 -hydroxyvalerate) and polycaprolactone

Colloidal lithography and embossing master are new techniques of producing nanotopography, which have been recently applied to improve tissue response to biomaterials by modifying the surface topography on a nano-scale dimension. A natural polyester (Biopol™), 8% 3-hydroxyvalerate-component (D400G) and a conventional biodegradable polycaprolactone (PCL) were studied, both nanostructured and native forms, in vitro and in vivo. Nanopits (100-nm deep, 120-nm diameter) on the D400G surface were produced by the embossing master technique (Nano-D400G), while nanocylinders (160-nm height, 100-nm diameter) on the PCL surface were made by the colloidal lithography technique (Nano-PCL). L929 fibroblasts were seeded on polyesters, and cell proliferation, cytotoxic effect, synthetic and cytokine production were assessed after 72 h and 7 days. Then, under general anesthesia, 3 Sprague–Dawley rats received dorsal subcutaneous implants of nanostructured and native polyesters. At 1, 4 and 12 weeks the animals were pharmacologically euthanized and implants with surrounding tissue studied histologically and histomorphometrically. In vitro results showed significant differences between D400G and PCL in Interleukin-6 production at 72 h. At 7 days, significant (P < 0.05) differences were found in Interleukin-1 β and tumor necrosis factor-α release for Nano-PCL when compared to Nano-D400G, and for PCL in comparison with D400G. In vivo results indicated that Nano-D400G implants produced a greater extent of inflammatory tissue than Nano-PCL at 4 weeks. The highest vascular densities were observed for Nano-PCL at 4 and 12 weeks. Chemical and topographical factors seem to be responsible for the different behaviour, and from the obtained results a prevalence of chemistry on in vitro data and nanotopography on soft tissue response in vivo are hypothesized, although more detailed investigations are necessary in this field.