Enhancing osteoconduction of PLLA-based nanocomposite scaffolds for bone regeneration using different biomimetic signals to MSCs.

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic "extracellular matrix"-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.

Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering.

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(epsilon-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 microm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell-material interaction are simultaneously requested.

Influence of different implant surfaces on peri-implant osteogenesis: histomorphometric analysis in sheep.

BACKGROUND: The present study investigated peri-implant osteogenesis and implant biologic fixation in different zirconia sandblasted endosseous titanium surfaces (SLA-60 and SLA-120) and a turned titanium surface (T) 2 and 4 weeks after surgery. METHODS: Seventy-two implant screws were implanted in tibia of six sheep. Histologic sections of implants (2 and 4 weeks after surgery) were analyzed with light microscopy for histomorphometric analysis of bone-to-implant contact (BIC), bone ingrowth (BI), and bone surface (BS/BV). Histologic blocks were used to perform bone microhardness studies next to the implants. Some implants were also observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: In general, the highest values of BIC, BI, BS/BV, and Vickers hardness number (HV) were measured in SLA-60 samples, followed by SLA-120 and T implants. Two weeks after surgery, all the implants appeared biologically fixed by a newly formed woven bone arranged in thin bone trabeculae and filling the gap between implant and host bone. Four weeks after implantation, the thickness of the woven bone trabeculae had increased, especially around the SLA-60 and SLA-120 implants by a gradual deposition of parallel-fiber bone. CONCLUSIONS: Our results suggest that, in the early period of peri-implant healing, the implant surface morphology that seemed to influence the increase of peri-implant osteogenesis, bone turnover, and peri-implant bone maturation was SLA-60. We suggest that this surface, characterized by moderately deep titanium cavities very similar to the osteocyte lacunae, could act as a microscopic scaffold for mesenchymal and/or osteoblast-like cells adhesion.

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. STATEMENT OF SIGNIFICANCE: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

Comparison of human mandibular osteoblasts grown on two commercially available titanium implant surfaces.

BACKGROUND: Surface characteristics play a major role in determining tissue response to implants and therefore their clinical outcome. The aim of the present study was to compare two commercially available titanium surfaces: plasma sprayed (TPS) and sand-blasted, acid-etched surface (SLA). METHODS: The surfaces were characterized by roughness testing, scanning electronic microscopy (SEM), Raman spectroscopy, and protein adsorption to determine their microtopographic and chemical properties. The effect of the surfaces on human mandibular osteoblasts was then studied in terms of cell morphology, adhesion, proliferation, and differentiation. Human osteoblasts from the mandible were cultured on these two surfaces and evaluated at 3, 6, 24, and 48 hours to determine cell attachment and morphology. Growth and differentiation kinetics were subsequently investigated by evaluating cell growth, alkaline phosphatase activity, osteocalcin and osteoprotegerin production at 7, 14, and 21 days. RESULTS: Although roughness was quite similar, the two surfaces presented strong differences in their topography, and cell morphology varied as a consequence. Osteoblasts on SLA appeared more elongated and spindle shaped than those on TPS, and their adhesion at 3 and 6 hours was weaker, but reached that of cells on TPS at hour 24. Cell proliferation was greater on SLA surfaces but differentiation parameters; i.e., alkaline phosphatase and osteocalcin, provided better results on TPS surfaces. Osteoprotegerin production was enhanced on TPS surfaces at days 14 and 21. CONCLUSION: Although cells grown on both surfaces exhibited good adhesion capabilities, a well-differentiated osteoblastic phenotype, and maintained a clear proliferation potential, our study suggests that plasma-sprayed treatment offers a better performance than SLA by creating, at least in the early phases, better conditions for tissue healing.

Different titanium surface treatment influences human mandibular osteoblast response.

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.

Osteogenesis and morphology of the peri-implant bone facing dental implants.

This study investigated the influence of different implant surfaces on peri-implant osteogenesis and implant face morphology of peri-implant tissues during the early (2 weeks) and complete healing period (3 months). Thirty endosseous titanium implants (conic screws) with differently treated surfaces (smooth titanium = SS, titanium plasma sprayed = TPS, sand-blasted zirconium oxide = Zr-SLA) were implanted in femur and tibiae diaphyses of two mongrel sheep. Histological sections of the implants and surrounding tissues obtained by sawing and grinding techniques were observed under light microscopy (LM). The peri-implant tissues of other samples were mechanically detached from the corresponding implants to be processed for SEM observation. Two weeks after implantation, we observed osteogenesis (new bone trabeculae) around all implant surfaces only where a gap was present at the host bone-metal interface. No evident bone deposition was detectable where threads of the screws were in direct contact with the compact host bone. Distance osteogenesis predominated in SS implants, while around rough surfaces (TPS and Zr-SLA), both distance and contact osteogenesis were present. At SEM analysis 2 weeks after implantation, the implant face of SS peri-implant tissue showed few, thin, newly formed, bone trabeculae immersed in large, loose, marrow tissue with blood vessels. Around the TPS screws, the implant face of the peri-implant tissue was rather irregular because of the rougher metal surface. Zr-SLA screws showed more numerous, newly formed bone trabeculae crossing marrow spaces and also needle-like crystals in bone nodules indicating an active mineralising process. After 3 months, all the screws appeared osseointegrated, being almost completely covered by a compact, mature, newly formed bone. However, some marrow spaces rich in blood vessels and undifferentiated cells were in contact with the metal surface. By SEM analysis, the implant face of the peri-implant tissue showed different results. Around the SS screws, the compact bone with areas of different mineralisation rate appeared very smooth, while around the rougher TPS screws, the bone still showed an irregular surface corresponding to the implant macro/microroughness. Around the Zr-SLA screws, a more regular implant-bone surface and sparse, calcified marrow spaces were detectable. Results from this research suggest that 2 weeks after implantation, trabecular bone represents the calcified healing tissue, which supports the early biological fixation of the implants. The peri-implant marrow spaces, rich in undifferentiated cells and blood vasculature, observed both 2 weeks and 3 months after surgery, favour the biological turnover of both early and mature peri-implant bone. The implant surface morphology strongly influences the rate and the modality of peri-implant osteogenesis, as do the morphology and arrangement of the implant face in peri-implant bone both during early healing (after 2 weeks) and when the implant is just osseointegrated; rough surfaces, and in particular Zr-SLA, seem to better favour bone deposition on the metal surface.

Influence of a zirconia sandblasting treated surface on peri-implant bone healing: An experimental study in sheep.

A sandblasting process with round zirconia (ZrO(2)) particles might be an alternative surface treatment to enhance the osseointegration of titanium dental implants. Our previous study on sheep compared smooth surface titanium implants (control) with implant surfaces sandblasted with two different granulations of ZrO(2). As the sandblasted surfaces proved superior, the present study further compared the ZrO(2) surface implant with other surface treatments currently employed: machined titanium (control), titanium oxide plasma sprayed (TPS) and alumina sandblasted (Al-SL) at different times after insertion (2, 4 and 12weeks). Twelve sheep were divided into three groups of four animals each and underwent implant insertion in tibia cortical bone under general anaesthesia. The implants with surrounding tissues were subjected to histology, histomorphometry, scanning electron microscopy and microhardness tests. The experimentation indicated that at 2weeks Zr-SL implants had the highest significant bone ingrowth (p<0.05) compared to the other implant surfaces, and a microhardness of newly formed bone inside the threads significantly higher than that of Ti. The present work shows that the ZrO(2) treatment produces better results in peri-implant newly formed bone than Ti and TPS processing, whereas its performance is similar to the Al-SL surface treatment.

The role of hydroxyapatite as solid signal on performance of PCL porous scaffolds for bone tissue regeneration.

Highly porous composites made up of biodegradable poly-epsilon-caprolactone (PCL) and stoichiometric hydroxyapatite (HA) particles have been developed as substrate for bone-tissue regeneration. The processing technique consists of phase inversion and particulate (salt crystals) leaching. Three different HA contents (13, 20 and 26 vol %) in PCL-based composite were considered in this study. Pore microstructure with fully interconnected network and pore sizes ranging around a few hundred of mum (macroporosity) was obtained as a result of salt particles removal by leaching process. Several microns (microporosity) porosity was also created through phase inversion of polymer solution. Total porosity up to 95% was achieved. Human marrow stromal cells (MSC) were seeded onto porous PCL-based composites for 1-5 weeks and cultured in osteogenic medium. MSC were able to adhere and grow on PCL-based substrates with a plateau at 3-4 weeks. However, the small effect of bioactive signals on the biological response evaluated in MSC cell culture suggests a prior role of topography on the biological response. Importantly, the presence of HA as a bioactive solid signal determines an increase of mechanical properties. On the overall, the results indicated that porous PCL-based composites are potential candidate for bone substitution with beneficial influence on structural characteristics by solid signal addition.