Nicolò Nicoli Aldini

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits.

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.

In vitro and in vivo behaviour of Ca- and P-enriched anodized titanium

The influence of different surface preparations on titanium biocompatibility and bone integration was evaluated. Commercially grade 2 titanium rods (diameter 2 mm, length: 3 mm), vacuum annealed and hydrofluoric acid etched was selected for its promising surface characteristics to achieve good direct osseointegration. Some rods were surface modified by Anodic Spark Discharge anodization and a thin layer (approximately 5 microm) of amorphous TiO2 containing Ca and P (Ti/AM) was obtained. Some of the Ti/AM specimens underwent a further hydrothermal treatment to produce a thin outermost layer (approximately 1 microm) of hydroxyapatite (Ti/AM/HA). Cytotoxicity tests (direct contact: ISO 10993-5) showed good cytocompatibility for all tested samples. Ti and tissue culture substrate + DMEM control, respectively, were associated with a significant higher proportion of attached cells than Ti/AM and Ti/AM/HA (P < 0.0005), but this was in the normal range of 10-20% of unattached cells for cytocompatible materials. Histomorphometric analysis conducted on samples inserted in the cancellous bone of distal femoral epiphysis of Sprague-Dawley rats gave the following results at 4 and 8 weeks: Affinity index (AI%) data proving the surface osteconductive properties of non-anodized acid etched Ti (AI-4 weeks: 67.1 +/- 17.0%; AI-8 weeks: 74.8 +/- 11.5%). Ti/AM samples showed the lowest values (AI-4 weeks: 45.8 +/- 15.9%; AI-8 weeks: 68.5 +/- 13.6%) while the best performances of the Ti/AM/HA samples (AI-4 weeks: 60.4 +/- 21.8%; AI-8 weeks: 79.5 + 9.37%) indicated that hydroxyapatite allowed a higher bone to implant contact respect to Ti only. Further investigations should be performed in order to better understand the mechanism of observed in vitro behaviour and to achieve information on long-term osseointegration process.

Stromal stem cells and platelet-rich plasma improve bone allograft integration.

Early vascular invasion is a key factor in bone allograft incorporation. It may reduce the complications related to slow and incomplete bone integration. Bone-marrow-derived stromal stem cells associated with platelet-rich plasma are potent angiogenic inducers proven to release vascular endothelial growth factor. Our goal was to test whether the combination of stromal stem cells and platelet-rich plasma is able to increase massive allograft integration in a large animal model with sacrifice at 4 months. A critical defect was made in the mid-diaphysis of the metatarsal bone of 10 sheep; the study group received an allograft plus stromal stem cells, platelet-rich plasma, and collagen (six animals) and the control group received only the allograft (four animals). Investigation was done with radiographs, mechanical tests and histomorphometric analysis, including new vascularization. Results showed substantial new bone formation in the allograft of the study group. Bone formation is correlated with better vascular invasion and remodeling of the graft in the study group. These results confirm the key role played by stromal stem cells and platelet-rich plasma in bone repair. Further studies are needed to better define the role stromal stem cells play when implanted alone.

Mechanical and histomorphometric evaluations of titanium implants with different surface treatments inserted in sheep cortical bone.

Improvement of the implant-bone interface is still an open problem and the interest in chemical modification of implant surfaces for cementless fixation has grown steadily over the past decade. Mechanical and histomorphometric investigations were performed at different times on implants inserted into sheep femoral cortical bone to compare the in vivo osseointegration of titanium screws ( X 3.5 x 7 mm length) with different surface treatments. After 8 weeks of implantation, the push-out force of anodized and hydrothermally treated implants (ANODIC) was significantly higher than that of machined implants (MACH) (36%, p<0.0005), whereas a decrease of 39% was observed for acid-etched implants (HF) when compared to other surface treatments. After 12 weeks of implantation, the push-out force values of HF implants were still significantly lower than those observed for MACH (-19%, p<0.01) and hydroxyapatite vacuum plasma-sprayed implants (HAVPS, -25%, p<0.0005), and the highest push-out force was found in HAVPS (p<0.001) implants. After 8 and 12 weeks of implantation, the AI of HF implants was significantly (p<0.05) lower ( approximately -25%) than that of MACH, HAVPS and ANODIC implants. In conclusion, results appear to confirm that there are no specific differences between ANODIC and HAVPS implants in terms of behavior. Moreover, although MACH implants show some surface contaminating agents, they appear to ensure good osseointegration within 12 weeks both mechanically and histomorphometrically, as do ANODIC and HAVPS implants. However, further studies are required to investigate bone hardness and mineralization around implants.

A bone substitute composed of polymethylmethacrylate and α-tricalcium phosphate: results in terms of osteoblast function and bone tissue formation

The biological properties of a composite polymeric matrix (PMMA + alpha-TCP) made of polymethylmethacrylate (PMMA) and alfa-tricalciumphosphate (alpha-TCP) was tested by means of in vitro and in vivo investigations. PMMA was used as a comparative material. Osteoblast cultures (MG 63) demonstrated that PMMA + alpha-TCP significantly and positively affected osteoblast viability, synthetic activity and interleukin-6 level as compared to PMMA. At 12 weeks, the PMMA + alpha-TCP implants in rabbit bone successfully osteointegrated in trabecular and cortical tissue (affinity index: 57.14+/-8.84% and 68.31+/-6.18%, respectively). The newly formed bone after tetracycline labelling was histologically observed inside PMMA + alpha-TCP porosity. The microhardness test at the bone-PMMA + alpha-TCP interface showed a significantly higher rate of newly formed bone mineralization compared with PMMA (+83.5% and +58.5%, respectively), but differences still existed between newly formed and pre-existing normal bone. It is herein hypothesized that the present positive results may be ascribed to the porous macroarchitecture of PMMA + alpha-TCP and the presence of the bioactive ceramic material that could have a synergic effect and be responsible for the improvement of (a) the material colonization by bone cells, (b) osteoblast activity, (c) osteoinduction and osteoconduction processes, (d) bone remodelling.

Effect of Mesenchymal Stem Cells and Platelet-Rich Plasma on the Healing of Standardized Bone Defects in the Alveolar Ridge: A Comparative Histomorphometric Study in Minipigs

PURPOSE The purpose of this study was to test the effect of the combination of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) incorporated into a fluorohydroxyapatite (FHA) scaffold on bone regeneration in cylindrical defects in the edentulous mandibular ridge of minipigs. MATERIALS AND METHODS Two mandibular premolar teeth were extracted bilaterally in 8 adult minipigs. After 2 months, 4 standardized defects of 3.5 mm diameter and 8 mm depth were created in each root site. The defects were randomly grafted with autogenous mandibular bone, FHA alone, PRP-FHA, or MSCs-PRP-FHA. A resorbable collagen membrane was placed over the defect area and the flaps were sutured. The animals were sacrificed 3 months later and biopsy samples were taken from the defect sites for histologic and histomorphometric assessment. RESULTS There was no evidence of inflammation or adverse tissue reaction with either treatment. MSCs-PRP-FHA-treated sites showed new vital bone between residual grafting particles. PRP-FHA- and FHA-treated sites showed residual particles in a background of marrow soft tissue with a moderate quantity of newly formed bone. Autogenous bone (46.97%) and MSCs-PRP-FHA (45.28%) produced a significantly higher amount of vital bone than PRP-FHA (37.95%), or FHA alone (36.03%). Further, the MSCs-PRP-FHA-treated defects showed a significantly higher percentage of contact between graft particles and newly formed bone compared with PRP-FHA and FHA group (59.23% vs 48.37% and 46.43%, respectively). CONCLUSIONS Our results suggest that, in this animal model, the addition of MSCs to PRP-FHA enhances bone formation after 3 months.

The effect of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study

Effects of pulsed electromagnetic fields (PEMFs, 75 Hz, 1.6 mT) were investigated in 12 rabbits after placing hydroxyapatite (HA) implants in their femoral condyles. Six animals were stimulated with PEMFs for three consecutive weeks, 6 h/day, while the remaining animals were sham‐treated (Control Group). Rabbits were sacrificed at 3 and 6 weeks (after a 3‐week non‐stimulation period) for histomorphometric analysis and microhardness testing (at 200, 500, 1000, 2000 μm from the implant) around the implants around the implants. Histomorphometric analysis did not highlight any significant changes. On the contrary, there were statistically significant differences between the effects produced by PEMFs and Control Groups (F = 149.70, p < 0.0005) on the Affinity Index results, as well as by the experimental time of 6 and 3 weeks (F = 17.12, p = 0.001) on the same results. In PEMF‐stimulated animals the microhardness (HV) values measured in trabecular bone at a distance of 200 and 500 μm from the implants, were significantly higher with respect to controls. At 6 weeks, HV values at the bone‐implant interface in PEMF‐stimulated animals were not significantly different with respect to normal bone, while they remained significantly lower in control animals. Both morphological and structural results demonstrated a positive therapeutic effect of PEMFs in accelerating HA osteointegration in trabecular bone.

Osteointegration of hydroxyapatite-coated and uncoated titanium screws in long-term ovariectomized sheep.

This study was designed to evaluate the osteointegration of HA-coated and uncoated titanium screws in the cortical bone of long-term (24 months) ovariectomized sheep (OVX group) compared to sham-aged sheep (Control group). At 12 weeks after implantation, the screws were tested biomechanically (extraction torque) and histomorphometrically (affinity index: AI) in both femoral and tibial diaphyses. Cancellous bone status was assessed by iliac crest biopsy. BMD of the L5 vertebra and a histomorphological study of the femoral and tibial shafts were performed to acquire data on cortical bone. A significant difference was found between the OVX and Control groups for BMD (p<0.0005), and a significant reduction in the cancellous bone area was observed in the OVX group. Femoral and tibial cortical bone parameters showed significant differences between the groups. The type of material selected (femurs: p<0.0005; tibiae: p<0.0005) and ovariectomy (femurs: p<0.005; tibiae: p<0.005) had a significant effect on the extraction torque. AI results were related to the presence or absence of ovariectomy (p<0.05) and strictly depended on the material implanted in the femur and tibia (p<0.0005). In conclusion, at implantation OVX sheep showed a significant loss of trabecular and cortical bone versus sham-aged sheep. The biomechanical and histomorphological results achieved suggest employing HA-coated screws in the presence of osteopenic cortical bone.

Dose-dependent effect of adipose-derived adult stem cells on vertical bone regeneration in rabbit calvarium.

Previous in vivo studies have shown a limited potential for vertical bone regeneration using osteoconductive scaffolds alone. In the present study, we investigated whether the association of adipose-derived adult stem cells (ASCs) with anorganic bovine bone (ABB) scaffold improved bone formation and implant osseointegration in a vertical guided bone regeneration model. Two pre-formed titanium domes were placed on the calvaria of 12 rabbits. Four treatment modalities were evenly distributed among the 24 domes: ABB alone, and ABB containing 3 x 10(5), 3 x 10(6), or 3 x 10(7) cells/graft. After 1 month, the domes were removed and one titanium implant was placed into each augmented site. One month after the second operation, the animals were killed and biopsy specimens were examined by histomorphometric and micro-CT analyses. Results indicated that at all concentrations, the ASC-loaded groups showed significantly more new bone formation and higher mean values of bone-implant contact and bone density inside threads than the ABB group. Furthermore, ASCs demonstrated a dose-response relationship, with the highest dose chosen inducing more robust bone regeneration. This study suggests that the delivery of ASCs on ABB might effectively increase vertical bone regeneration and implant osseointegration, versus ABB alone.

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.