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.

Long-term in vivo experimental investigations on magnesium doped hydroxyapatite bone substitutes.

Despite several efforts to find suitable alternatives to autologous bone, no bone substitute currently available provides the same characteristics and properties. Nevertheless, among the wide range of materials proposed as bone substitutes, calcium phosphate materials represent the most promising category and the present study is aimed at improving the knowledge on non-stoichiometric magnesium-doped hydroxyapatite substitutes (Mg-HA), tested in two different formulations: Mg-HA Putty and Mg-HA Granules. These bone substitutes were implanted bilaterally into iliac crest bone defects in healthy sheep and comparative histological, histomorphometric, microhardness and ultrastructural assessments were performed 9, 12, 18 and 24 months after surgery to elucidate bone tissue apposition, mineralization and material degradation in vivo. The results confirmed that the biomimetic bone substitutes provide a histocompatible and osteoconductive structural support, during the bone formation process, and give essential information about the in vivo resorption process and biological behavior of biomimetic bone substitutes.

Effects of different crosslinking conditions on the chemical-physical properties of a novel bio-inspired composite scaffold stabilised with 1,4-butanediol diglycidyl ether (BDDGE).

Serious cartilage lesions (Outerbridge III, IV) may be successfully treated with a three-layered gradient scaffold made by magnesium-doped hydroxyapatite and type I collagen, manufactured through a bio-inspired process and stabilised by a reactive bis-epoxy (1,4-butanediol diglycidyl ether, BDDGE). Each layer was analysed to elucidate the effects of crosslinking variables (concentration, temperature and pH). The chemical stabilisation led to an homogeneous and aligned collagenous matrix: the fibrous structures switched to a laminar foils-based arrangement and organic phases acquired an highly coordinated 3D-organization. These morphological features were strongly evident when crosslinking occurred in alkaline solution, with BDDGE concentration of at least 1 wt%. The optimised crosslinking conditions did not affect the apatite nano-crystals nucleated into self-assembling collagen fibres. The present work allowed to demonstrate that acting on BDDGE reaction parameters might be an useful tool to control the chemical-physical properties of bio-inspired scaffold suitable to heal wide osteochondral defects, even through arthroscopic procedure.

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.

Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses.

The present in vivo preliminary experiment is aimed at testing mechanical and biological behaviour of a new nano-structured composite multilayer biomimetic scaffold for the treatment of chondral and osteochondral defects. The three-dimensional biomimetic scaffold (Fin-Ceramica Faenza S.p.A., Faenza-Italy) was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles, in two configurations, bi- and tri-layered, to reproduce, respectively, chondral and osteochondral anatomy. Chondral defects (lateral condyle) and deep osteochondral defects (medial condyle) were made in the distal epiphysis of the third metacarpal bone of both forelimbs of two adult horses and treated respectively with the chondral and osteochondral grafts. Both animals were euthanised six months follow up. The images obtained at the second look arthroscopy evaluation, performed two months after surgery, demonstrated good filling of the chondral and osteo-chondral defects without any inflammatory reaction around and inside the lesions. At the histological analysis the growth of trabecular bone in the osteochondral lesion was evident. Only in one case, the whole thickness of the osteochondral lesion was filled by fibrocartilaginous tissue. The formation of a tidemark line was evident at the interface with the newly formed bone. Newly formed fibrocartilaginous tissue was present in the area of the chondral defect. Initial alignment of the collagen fibres was recognisable with polarised light in both groups. The results of the present pilot study showed that this novel osteochondral and chondral scaffold may act as a suitable matrix to facilitate orderly regeneration of bone and hyaline-like cartilage.

A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesions: technique note and an early stability pilot clinical trial.

INTRODUCTION: Osteochondral articular defects are a key concern in orthopaedic surgery. Current surgical techniques to repair osteochondral defects lead to poor subchondral bone regeneration and fibrocartilage formation, which is often associated with joint pain and stiffness. The objective of this pilot clinical study is to evaluate the performance and the intrinsic stability of a newly developed biomimetic osteochondral scaffold and to test the safety and the feasibility of the surgical procedure. METHODS: A gradient composite osteochondral scaffold based on type I collagen-hydroxyapatite was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. Thirteen patients (15 defect sites) were treated with scaffold implantation from January 2007 to July 2007: four at the medial femoral condyle, two at the lateral femoral condyles, five at the patellas and four at the trochleas. The mean size of the defects was 2.8 cm(2) (range: 1.5-5.9 cm(2)). All patients were followed up prospectively. High-resolution magnetic resonance imaging (MRI) was used to determine "the early postoperative adherence rate" at 4-5 weeks and 25-26 weeks after scaffold implantation. Moreover, the magnetic resonance observation of cartilage repair tissue (MOCART) score was performed on every MRI. Two second-looks were performed at 6 months; cartilage repair was assessed using the International Cartilage Repair Society (ICRS) visual scoring system and histological and immunohistochemical analysis of the two biopsies was carried out. RESULTS: A completely attached graft and repair tissue were found in 13 of 15 lesions (86.7%). A partial detachment was observed in two patients (13.3%). No detached grafts were found. Complete filling of the cartilage defect and congruency of the articular surface were seen in 10 lesions (66.7%) with MRI evaluation at 6 months. The complete integration of the grafted cartilage was detected in eight lesions (53.3%). Subchondral bone changes (oedema or sclerosis) were found in eight defects (53.3%). Statistical analysis showed a significant improvement in the International Knee Documentation Committee (IKDC) subjective and objective scores from preoperative to 6 months' follow-up (p<0.0005). Visual scoring of the repaired tissue at second-look revealed a normal repair score in one case and a near-normal repair score in the other case. Histological analysis showed the formation of subchondral bone without the presence of biomaterial. The cartilage repair tissue appeared to be engaged in an ongoing maturation process. CONCLUSIONS: The technique is safe and MRI evaluation at short-term follow-up has demonstrated good stability of the scaffold without any other fixation device. The preliminary clinical results at short-term follow-up are encouraging. A clinical and MRI study with longer follow-up and randomised studies will be done to confirm the high potential of this novel osteochondral scaffold.

Adhesion reduction after knee surgery in a rabbit model by Hyaloglide, a hyaluronan derivative gel.

A randomized controlled experimental trial was performed in a rabbit model of surgical adhesions to investigate the anti-adhesive effects of Hyaloglide, a highly viscous hyaluronan derivative absorbable gel after knee surgery. Twenty New Zealand white rabbits were prepared and randomly divided into two groups of 10 animals each. An intra-articular fibro-adhesive scar was created in the right knee joint of the hind paw of each rabbit using a standardized surgical procedure, and Hyaloglide was administered into the joint cavity of the knee at the end of intervention in the animals belonging to the treatment group. No anti-adhesive treatment was applied in the control group. Additionally, immobilization using a Kirschner wire was applied in order to increase the risk of adhesions. Six weeks after surgery the animals were euthanized and after removal of the immobilization system, adhesions were evaluated both macroscopically and histologically. Results of gross observations using a specific adhesion scoring system showed a significant reduction (p<0.01) of both incidence and severity of adhesions in the hyaluronan-treated group compared to the control group. Histologically, adhesions in the treated group were thinner with less collagenic fibers. In conclusion, Hyaloglide may be considered as a promising absorbable barrier for prevention of post-operative fibrotic adhesions after knee surgery.

Prevention of postsurgical adhesions with an autocrosslinked hyaluronan derivative gel.

BACKGROUND: ACP gel is a new crosslinked derivative of hyaluronic acid (HA) that displays the biocompatibility properties of its original polymer but has a higher viscosity. It has been demonstrated in an animal model that the gel reduces adhesions after gynecological surgery. The aim of the present study was therefore to investigate the efficacy of ACP gel in increasing viscosity for the prevention of adhesions after abdominal surgery. METHODS: The antiadhesive effect of ACP gel was tested in a controlled randomized study using a standardized animal model of abdominal surgery involving the creation of defects in the parietal peritoneum and muscular fascia and cecal abrasion. The animals (100 female New Zealand white rabbits) were randomly allocated into five treatment groups to receive: ACP gel (1, 2, 4, and 6%) on the injured area or no ACP gel (control). The incidence of adhesions and their grade (score 0-11) were blindly evaluated 10 weeks after surgery. RESULTS: The percentages of adhesion-free animals were 60, 84, 90, and 84% in the 1, 2, 4, and 6% ACP gel concentration groups, respectively, versus 15% in the control group (P = 0.001). The mean adhesion scores were 3.00 +/- 0.91, 1.37 +/- 0.75, 0.65 +/- 0.45, and 1.16 +/- 0.64 in the 1, 2, 4, and 6% ACP gel groups, respectively, versus 7.70 +/- 0.83 in the control group (P < 0.001). CONCLUSION: ACP gel prevents postsurgical abdominal adhesions even at a 1% concentration. This finding may be of clinical importance in situations in which large volumes of antiadhesive solution are required.

Efficacy of a hyaluronan derivative gel in postsurgical adhesion prevention in the presence of inadequate hemostasis.

BACKGROUND: We previously demonstrated that an auto-cross-linked hyaluronan-based antiadhesion agent (auto-cross-linked polysaccharide [ACP] gel) was effective in postsurgical adhesion prevention after open laparotomy and laparoscopic surgery with adequate hemostasis in animal models. This study assessed the ability of different preparations of ACP gel to prevent adhesions in the presence of bleeding or inadequate hemostasis. METHODS: Ninety-seven female rabbits were subjected to a standardized surgical lesion with subsequent exudative abdominal bleeding (oozing model), and 97 animals were subjected to a standardized surgical lesion with severe abdominal bleeding (bleeding model). After injury, the animals were randomly assigned to 5 groups of treatment: 3 different preparations of ACP gel (20, 40, and 60 mg/mL), a hyaluronan-carboxymethylcellulose film, and no treatment. Three weeks after operation, the animals were killed, and the adhesions were assessed by a blinded observer who measured the length and area of the adhesions and who used the Blauer scoring system. RESULTS: All 3 preparations of ACP gel and the hyaluronan-carboxymethylcellulose film reduced adhesion formation in both models (P <.01) as measured by the number of adhesion-free animals, mean Blauer score, and the mean length and surface area of the adhesions. There were no statistical differences between the different treatment groups. CONCLUSIONS: These data suggest that different hyaluronan based agents in the presence of severe bleeding or exudative abdominal bleeding reduce de-novo postsurgical adhesion formation.

A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model.

OBJECTIVE: To evaluate the effectiveness of a crosslinked hyaluronan solution (ACP gel) in the prevention of postsurgical adhesions in laparoscopic surgery. DESIGN: A randomized blinded study using a rabbit model in laparoscopic surgery. SETTING: A standardized surgical trauma in the rabbit uterine horn to induce adhesion formation. ANIMALS: Sixty-four sexually matured female New Zealand white rabbits weighing 2.5 to 3.0 kg and aged 3-4 months. INTERVENTION(S): After trauma, group 1 (n = 22) received no treatment, group 2 animals (n = 20) received oxidized-regenerated cellulose (Interceed [TC7]) in group 3 (n = 22) 5 mL of ACP gel were applied on the lesion. MAIN OUTCOME MEASURE(S): Six weeks after laparoscopy, a laparotomy was performed and the adhesions were scored according to Blauer's scoring system. RESULT(S): 66% of the untreated animals and 85% of the animals treated with Interceed presented with severe adhesions, whereas only 35% of the ACP gel treatment group had significant adhesions. The mean ( +/- SEM) increased adhesion score was 2.24 +/- 0.26 in the untreated group, 2.45 +/- 0.22 in the Interceed group, and was 1.25 +/- 0.28 in the ACP gel group. CONCLUSION(S): This study revealed that ACP gel holds promise as a novel resorbable biomaterial for the reduction of postoperative adhesions after laparoscopic surgery.