Fracture nonunion is a severe clinical problem for the patient, as well as for the clinician. About 5-20% of fractures does not heal properly after more than six months, with a 19% nonunion rate for tibia, 12% for femur and 13% for humerus, leading to patient morbidity, prolonged hospitalization, and high costs.

The standard treatment with iliac crest-derived autologous bone filling the nonunion site may cause pain or hematoma to the patient, as well as major complications such as infection.

The application of mesenchymal autologous cells (MSC) to improve bone formation calls for randomized, open, two-arm clinical studies to verify safety and efficacy.

The ORTHOUNION * project (ORTHOpedic randomized clinical trial with expanded bone marrow MSC and bioceramics versus autograft in long bone nonUNIONs) is a multicentric, open, randomized, comparative phase II clinical trial, approved in the framework of the H2020 funding programme, under the coordination of Enrique Gòmez Barrena of the Hospital La Paz (Madrid, Spain).

Starting from January 2017, patients with nonunion of femur, tibia or humerus have been actively enrolled in Spain, France, Germany, and Italy.

The study protocol encompasses two experimental arms, i.e., autologous bone marrow-derived mesenchymal cells after expansion (‘high dose’ or ‘low dose’ MSC) combined to ceramic granules (MBCP™, Biomatlante), and iliac crest-derived autologous trabecular bone (ICAG) as active comparator arm, with a 2-year follow-up after surgery.

Despite the COVID 19 pandemic with several lockdown periods in the four countries, the trial was continued, leading to 42 patients treated out of 51 included, with 11 receiving the bone graft (G1 arm), 15 the ‘high dose’ MSC (200x10⁶, G2a arm) and 16 the ‘low dose’ MSC (100x10⁶, G2b arm).

The Rizzoli Orthopaedic Institute has functioned as coordinator of the Italian clinical centres (Bologna, Milano, Brescia) and the Biomedical Science and Technologies and Nanobiotechnology Lab of the RIT Dept. has enrolled six patients with the collaboration of the Rizzoli’ 3rd Orthopaedic and Traumatological Clinic prevalently Oncologic.

Moreover, the IOR Lab has collected and analysed the blood samples from all the patients treated to monitor the changes of the bone turnover markers following the surgical treatment with G1, G2a or G2b protocols.

The clinical and biochemical results of the study, still under evaluation, are presented.

* ORTHOUNION Horizon 2020 GA 733288

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and bone inducers, is a potential alternative to conventional treatments. Pre-clinical testing of innovative scaffolds relies on in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs) is required to mimic their crosstalk and molecular cooperation for bone remodelling. To this aim, two composite materials based on type I collagen were developed, containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. Following chemical crosslinking with genipin, the nanostructured materials were tested for 2–3 weeks with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors. The favourable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of bone cells, encouraging a further investigation of the two bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

Bone tissue engineering is a promising strategy to treat the huge number of bone fractures caused by progressive population ageing and diseases i.e., osteoporosis. The bioactive and biomimetic materials design modulating cell behaviour can support healthy bone tissue regeneration. In this frame, type I collagen and hydroxyapatite (HA) have been often combined to produce biomimetic scaffolds. In addition, mesoporous bioactive glasses (MBGs) are known for their ability to promote the deposition of HA nanocrystals and their potential to incorporate and release therapeutic ions. Furthermore, the use of 3D printing technologies enables the effective design of scaffolds reproducing the natural bone architecture.

This study aims to design biomimetic and bioactive 3D printed scaffolds that mimic healthy bone tissue natural features in terms of chemical composition, topography and biochemical cues. Optimised collagenous hybrid systems will be processed by means of extrusion 3D printing technologies to obtain high resolution bone-like structures. Protocols of human co-cultures of osteoblasts and osteoclasts will be developed and used to test the 3D scaffolds.

Type I collagen has been combined with rod-like nano-HA and strontium containing MBGs (micro- and nano-sized particles) in order to obtain hybrid systems resembling the composition of native bone tissue. A comprehensive rheological study has been performed to investigate the potential use of the hybrid systems as biomaterial inks. Mesh-like structures have been obtained by means of extrusion-based technologies exploiting the freeform reversible embedding of suspended hydrogels (FRESH) approach. Different crosslinking methods have been tested to improve final constructs mechanical properties. Both crosslinked and non-crosslinked biomaterials were cultured with human osteoblasts and osteoclasts to assay the hybrid matrix biocompatibility as well as its influence on cell behaviour.

Homogeneous hybrid systems have been successfully developed and characterised, proving their suitability as biomaterial inks for 3D printing technologies. Mesh-like structures have been extruded in a thermo-reversible gelatine slurry, exploiting the sol-gel transition of the systems under physiological conditions. Covalent bonds between collagen molecules have been promoted by genipin treatment, leading to a significant increase in matrix strength and stability. The collagen methacrylation and the further UV-crosslinking are under investigation as alternative promising method to reinforce the 3D structure during the printing process. Biological tests showed the potential of the developed systems especially for genipin treated samples, with a significant adhesion of primary cells.

Collagenous hybrid systems proved their suitability for bioactive 3D printed structures design for bone tissue engineering. The multiple stimuli provided by the scaffold composition and structure will be investigated on both direct and indirect human osteoblasts and osteoclasts co-culture, according to the developed protocols.

Osteoporosis is a worldwide disease with a high prevalence in elderly population; it results in bone loss and decreased bone strength that lead to low-energy fractures. Since antiresorptive treatments could lead to long-term adverse effects, the ERC BOOST project aims to propose a biomimetic 3D-printed scaffold reproducing the architecture and chemistry of healthy bone. In this study, the structural parameters of healthy bone were studied in order to reproduce them through 3D printing; furthermore, structural and mechanical differences between healthy and osteoporotic (OP) bones were assessed. Healthy and OP humeral heads discarded during surgical interventions (following ethical approval by Istituto Ortopedico Rizzoli-Italy) were tomographically analysed to obtain bone structural parameters. Successively, 8 mm diameter biopsies were harvested from the heads and underwent compression and nanoindentation tests to investigate macroscopic and microscopic mechanical properties, respectively. XRD measurements were performed on bone fragments. OP bone samples exhibited inferior mechanical properties to their less interconnected and more anisotropic structure, with thinner trabeculae and larger pores. On the other hand, nanoindentations performed on OP trabeculae showed increased Young Modulus compared to healthy samples probably due to their increased hydroxyapatite crystal size, as revealed by XRD. Osteoporosis causes the weakening of the trabecular structure that leads to a decrease of bone mechanical properties. However, OP trabeculae are stiffer due to increased dimensions of hydroxyapatite crystals.

Background

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

The success of biomaterials lies in the direct interaction with the host tissue. Calcium phosphates (CaP) stand as an alternative graft material for bone regeneration due to their similar composition to natural bone. Few studies have focused on the early stages of bone-like material remodeling by osteoclasts (OC), though the CaP fate is to be resorbed and then replaced by new bone. Instead, to understand how osteoclasts modify the CaP surface and initiate resorption, so as to influence subsequent osteoblast activities and bone formation, is mandatory.

Sintered hydroxyapatite (s-HA) and biomimetic hydroxyapatite with two different microstructures (b-HA-C, coarse and b-HA-F, fine) discs (1500×250 µm²) were produced from the same reagents [1]. Tissue culture polystyrene (TCPS) was used as control. Precursor human OC from buffy coats were seeded on ceramic substrates [6·10⁶cells/cm²] and supplemented with RANKL-containing osteoblast supernatant as differentiation medium over 21 days. Cell interaction with the biomaterials was investigated in terms of OC adhesion and differentiation, with gene expression, tartrate-resistant acid phosphatase (TRAP) and Hoechst staining for OC maturation. Cell culture supernatants were analyzed for ionic exchange, namely Ca and P, due to biomaterials or cells. Osteoclasts morphology was evaluated using SEM at 21 days. Innovatively, focused ion beam (FIB) was used to evaluate biomaterial structure beneath the OC to further investigate the resorption effects. To this aim, selected OC were cut cross-sectioned using a Gallium ion beam at an acceleration of 30KV, followed by a coarse milling at 10nA and a deposition of platinum to achieve a fine milling at 500pA.

Clear differences in cellular behavior were noted relative to the different substrate microstructures. Control TCPS and s-HA showed similar TRAP-positive staining and gene expression for mature OC. Several resorption pits with partial dissolution of the equiaxial grains of s-HA were noticed. b-HA substrates also showed attached and differentiated TRAP-positive OC, but gene expression resulted lower than control and s-HA. However, morphological evaluation with SEM-FIB interestingly showed early stages of osteoclast-mediated degradation on b-HA-F, i.e.an increased surface roughness in the substrate underlying cells. B-HA-C also showed attached and mature OC with a scarce degradation activity

FIB technique has been applied to cell-seeded CaP and shown as a viable method to investigate OC morphology and resorption. Though gene expression showed similarities for both biomimetic substrates, substrate morphology observed underneath OC was significantly different. b-HA-F showed early stages of OC mediated degradation underneath well spread cells similar to those seen on s-HA. No resorptive activity was found on b-HA-C even though gene expression values were similar to b-HA-F: both the acute ion exchange and the surface tortuosity on b-HA-C could explain the difficulty with the resorptive process by OC. In conclusion focused ion beam technique complements SEM imaging and may disclose changes in the inner structure of materials due to cell/material interactions.

The regenerative potential of bone grafts is tightly linked to the interaction of the biomaterial with the host tissue environment. Hence, strategies to confer artificial extracellular matrix (aECM) cues on the material surface are becoming a powerful tool to trigger the healing cascade and to stimulate bone regeneration. The use of glycosaminoglycans (GAGs), such as heparin, as aECM components has gained interest in the last years as a strategy to improve biological response. Calcium phosphates (CaP) are extensively used as bone grafts, however no studies have investigated the effect of GAG functionalisation on their surface. Some authors have focused on the effects of GAGs on osteoblastic cells, however, little work has been performed on the interaction with osteoclasts (OC), and still the reported effects are controversial [1]. The aim of this study was to investigate the effect of heparin on osteoclastic fate in terms of adhesion and differentiation.

Sintered CaP (β-TCP) and biomimetic CaP (calcium-deficient hydroxyapatite, CDHA) discs were synthesized at 1100 ºC and at 37ºC, respectively. Heparinisation was achieved though silane coupling (APTES) followed by amidation in the presence of EDC/NHS to covalently link heparin. The osteoclast response of heparinised (H) vsnon-heparinised substrates was studied using human monocytes as OC precursors. Tissue culture plastic (TCPS) was used as a control sample. Cell densities were 6·10⁶and 3·10⁶cells/cm²for biomaterials and TCPS, respectively. Cell cultures were supplemented every 3 days with 25% supernatant of osteoblast-like cell line as a source of RANKL, as well as other stimulating factors [2]. Tartrate-resistant acid phosphatase and Hoechst staining were used to evaluate OC adhesion, differentiation and morphology at different time points from seeding on the surfaces (14–21–28 days).

OC precursors showed adhesion on all substrates. β-TCP and β-TCP-H hosted higher number of OC precursors which might be related to the smoother sintered surface of the materials. Oppositely, the high roughness of CDHA and CDHA-H hamper the adhesion of OC, hence a lower number of cells was observed on heparin-coated and uncoated biomimetic apatites. However, the maturation of OC precursors was found to take place at earlier times (14days) on biomimetic substrates compared to sintered ones. TCPS, CDHA, CDHA-H and β-TCP-H showed clearly differentiated OC at 14 days, as revealed by TRAP positivity and multinuclearity. Interestingly, CDHA-H and β-TCP-H induced the highest multinuclearity among all differentiated OC. Both heparinised substrates point at an enhancing effect of heparin on OC maturation.

OC precursors are able to differentiate on β-TCP and CDHA substrates, a process enhanced when heparin functionalisation is performed on the materials surface. In our hands heparinisation is promoting OC differentiation at early time points, similarly to TCPS control. Interestingly, heparin substrates induced larger TRAP positive-OC and higher multinuclearity in the mature OC than TCPS control. As pointed out by Irie et al., heparin might interact through the RANKL/OPG ratio [3], thus inhibiting OPG activity and enhancing RANKL which triggers OC maturation.

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. The REBORNE project entailed a multi-center orthopaedic clinical trial focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial, to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

Peripheral blood was collected from patients at fixed time-endpoints, that is at 6,12 and 24 weeks post-surgery for implantation of expanded autologus MSC and bone-like particles. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-up visits.

A significant relationship with age was found only at 6 months, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the baseline value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with a negative outcome. The BTM profile apparently indicated a remarkable bone formation activity 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome.

Blood markers related to bone cell function are useful to measure the efficacy of a expanded MSC-regenerative approach applied to long bone non-unions. Changes of the markers may provide a support to radiological assessment of bone healing.

Ceramic-on-ceramic bearing is an attractive alternative to metal-on-polyethylene bearing due to the unique tri-bological advantages of alumina. However, despite the long-term satisfactory results obtained so far in the vast majority of patients, failure may occur in a few cases.

Clinical, radiographic, laboratory and microbiological data of 30 consecutive subjects with failed alumina-on-alumina total hip arthroplasties (THA) were analyzed to define if foreign body reaction to wear debris may be responsible for periprosthetic bone resorption, as in conventional metal-to-polyethylene bearings. In all cases, clinical and radiographical material was reviewed, retrieved implants were examined, and histology of periprosthetic tissues was analyzed. Massive osteolysis was never observed. Apart from 5 five patients for which revision surgery was necessary due to the occurrence of late infection, in all other cases failure had occurred due to secondary implant instability (as in the case of screwed sockets, 19 cases) or to malpositioning of the implant (5 cases). One patient suffered from chronic dislocation.

In the vast majority of cases, ceramic wear debris was absent or scarce, and did not induce any tissue reaction. In a few cases with severe wear, debris was evident in clusters of perivascular macrophages, notably in the absence of foreign body multinucleated cells, confirming the excellent biocompatibility of ceramics.

These findings indicate that wear debris and peri-prostetic bone resorption were the effect rather than the cause of failure, differently from revised metal-on-polyethylene bearings, in which foreign body cell reaction is the main pathogenetic mechanism of failure. On the contrary, mechanical problems, due to incorrect surgical technique or to inadequate prosthetic design, may cause instability of the implant, in turn resulting in wear debris production and moderate if any biological reaction.

Aims: Surface modification of biomaterials to be used as scaffolds in tissue engineering is a promising method to improve device multi-functionality and biological properties. Biomimetic surface treatments, such as changes in nano-structure and attachment of biomolecular signals, enhance material bioactivity and affinity for specific cells. In this study the functionalization of a titanium surface with vitronectin-derived nonapeptide(HVP) and RGD peptides was investigated. Bone forming cells were used to analyse the role of each surface modification in the initial steps of cell adhesion process and then proliferation and differentiation.

Method: Smooth titanium samples were functionalized by different chemical treatments in order to obtain varying amount of peptide adhesion. Human marrow stro-mal cells (MSC) were seeded and cultured in osteogenic medium. Cell adhesion and morphology were assessed by fluorescence microscopy after 6 hours. Viability of MSC was quantified at 7 and 14 days from plating, proliferation was measured using DNA and total protein content, and osteoblast phenotype expression was assayed using alkaline phosphatase (ALP) and calcium content.

Results: The results showed that presence of HVP and RGD peptides improves cell morphology in early adhesion on surface, compared to control (titanium without peptides). Activity of ALP and Ca²⁺ content of (1:1000)HVP and RGD samples were higher than the other experimental surfaces with or without peptides, even if they did not reach the values of control cells on tissue culture polystyrene.

Discussion: Properties acquired with chemical treatments can improve titanium surfaces. These data provide information useful to develop biomimetic cell-friendly surfaces for bone engineering.

Aims: It is well known that the success of an orthopedic implant is determined by a close apposition between bone and implant surface. The excellent physical properties and the controlled degradation of poly-ε-caprolactone (PCL) has been shown, however the suitability for bone engineering applications of a material is critically influenced by the interactions between cells and scaffold. The aim of this study was to evaluate the interaction between bone marrow cells and PCL surface. Bone marrow cells were obtained from femurs of New Zealand rabbits and seeded on PCL directly (WBMC) or after gradient centrifugation (MSC), mimicking the in vivo colonization of PCL after implantation and the pre-seeding strategy.

Methods: PCL was dissolved in chloroform (3% w/v solution) and spin coated as a thin (100nm) film onto p-doped silicon wafers. The surface wettability and roughness were analyzed by SFE measurements and AFM. Cells were seeded on PCL and adhesion/proliferation evaluated at 1, 7, 14, 21 and 28 days. Fluorescence microscopy and SEM imaging were performed at defined time endpoints.

Results: At 2 wks adherence-selected MSC had already formed confluent multilayers, whereas WBMC were still semi-confluent. At 4 wks a consistent layer of ECM was observed underneath the cell layers of both cultures.

Conclusions: PCL is a proper substrate for bone cell attachment and growth, as cell confluence was reached at 2 wks for MSC and at 3–4 wks for WBMC. To avoid any risk of bacterial contamination, the seeding of WBMC on PCL scaffold, which implies reduced handling of cells outside the body, was shown to be effective and may be recommended in the clinical practice.

Aims: Different polyurethane foam matrices (PUF) loaded with hydroxyapatite (HA) or α-tricalcium phosphate were proposed as scaffold for bone regeneration [1,2]. In this work new PUFs were developed and loaded with HA or α-TCP.

Methods: PUFs were synthesized by a one-step polymerisation from a hydrophilic polyol mixture (LF 2946, Elastogran, Italy) and polymeric MDI (B141, BASF), using Fe acetyl-acetonate as catalyst and 2% water as expanding agent. The composites were prepared in the same way, by adding HA or α-TCP to the reacting mixture.

In vitro cell interactions were evaluated with human osteoblasts (HOB, 2nd passage) isolated from the trabecular bone of the femoral head of patients undergoing total hip replacement and cultured following the usual procedure. HOB cells (1x105 cells/sample) were kept in contact with the scaffolds for 7 and 14 days. At each time endpoint HOB metabolic activity, intracellular and released ALP were evaluated.

Results: By water adsorption test, newly synthesized PUFs showed a higher hydrophilicity compared to that of the previous matrices (600% vs 110% water uptake after 100h). Due to the presence of inorganic salts, composite scaffolds showed density values higher (0.131B80.200g/cm3) than those of unloaded PUFs (0.071B80.093g/cm3). Yet, open cell percentage (57–75%) and average pore size (350B8520mm) resulted similar to those of the PUFs.

HOB cells grown on scaffold samples showed an increase of metabolic activity from 7 to 14 days. The amount of intracellular ALP increased too, whereas the amount of ALP in the medium was quite low. HOB cells, after 14 days, appeared closely adherent to the scaffolds, with an elongated and flattened shape.

Conclusions: These preliminary results showed that, even if slow, the growth of HOB onto PUF scaffolds was quite good. After 14 days, PUF composites showed higher cells growth than PUF-matrices, confirming the role of the bone-like inorganic particles in improving osteoblasts functions. Long-term in vitro tests are now in progress.

The placement of orthopaedic, as well as dental, oral and craniofacial implants, are common practices in medicine and denstistry today. Challenges to the successful outcome of such implants include loosening of the device and inadequate filling of bone defects. The engineering of bone tissue is a recent strategy to provide new solutions to such problems. Since skeletal tissue regeneration requires three components, i.e. cells, growth and differentiation factors, and extracellular matrix, the approach of bone engineering is to mimic the biological process by delivering to the injured site: 1. cells capable of differentiating into osteoblasts, 2. inductive factors, and 3. a scaffold, biodegradable or not, to support cells. Prior to experimental and clinical application of the innovative surfaces or scaffolds, the three components have to be tested in the Labs using reliable in vitro methods.

1. Cells. The source of cells is a key point: osteoblast is the differentiated cell able to form bone in vivo and in vitro, and should be used, but primary human osteoblasts (hOB) are seldom available to the Labs, whereas osteoblast-like cell lines and bone cells from animals are an easy source, but may give different responses. An additional aspect which cannot be disregarded is the source of the bone cells, since the age and gender of the donor, as well as the site of retrieval and the method of isolation, have been shown to affect the yield of cells, the proliferation rate and their ability to form bone in vitro.

Stromal cells from bone marrow (MSC), and other sites, have been shown to be a promising source of cells with high replicative and bone-forming potential. The same drawbacks outlined for osteoblasts apply to MSC.

In our lab human osteoblasts are mainly obtained from trabecular bone fragments and stromal cells from bone marrow of patients undergoing surgical revision of hip implants. HOB are usually isolated by seeding minced bone chips in culture plates to get outgrowth of single cells from fragments, as the isolation technique (mechanical vs enzymatic) appeared to have no effect on the differentiation process. Confluence of the cell layer is reached in approximately four weeks (14–40 dd) and the bone phenotype is assessed by alkaline phosphatase (ALP) cytochemistry and morphology, as well as mineralization after addition of ascorbic acid and b-glycerophosphate. MSC are isolated by gradient centrifugation and adherence to culture plastic; their replicative potential is evaluated by the colony forming assay, and ALP staining provides the test for differentiation toward bone-forming cells.

Preliminary evaluation of our cell isolates from orthopaedic patients showed that there is no direct correlation between the age of donor and the yield of hOB in terms of proliferation rate and ALP activity. As far as MSC are concerned, the addition of dexamethasone during cell expansion stimulated only a small increase in the number of colonies and ALP positive staining.

2. Inductive factors include growth factors, cytokines, peptide sequences and angiogenetic factors. The experience of our Lab will be given in a different presentation.

3. Specifically tailored biomaterials are crucial tools in tissue engineering: our experience is concerning in vitro testing of artificial materials developed by material scientists to replace bone.

Such materials have to provide biocompatibility, i.e. no inflammatory reaction or immunorejection, controlled biodegradation if necessary, and biomechanical features to comply with the anatomical requirements.

From a methodological point of view, the ‘engineered’ biomaterials can be classifieded as bi-dimensional (2D) materials or three-dimensional (3D) scaffolds.

2D surfaces are often well known materials already in clinical use, but innovations concern the ‘biomimetic approach’ applied to their surface. This means to recreate the ‘nanotopography’ of natural tissues, by modifying the roughness, or by mimicking the extracellular matrix (ECM) on the surface: both strategies aim to recruit bone cells and to promote bone formation.

In the framework of a national research project both 2D and 3D materials were assayed in our Lab.

Two types of titanium with different surfaces were tested with human osteoblasts, and compared to a commercial titanium with smooth surface. At 4 hours from seeding onto surfaces, hOB on smooth Ti were elongated, with evident spreading. On the rougher surfaces small focal contact patches were evident, and hOB showed a more rounded morphology whereas stitching to the irregular surface. By prolonging the culture time, all the surfaces were covered by cells, and differences were less evident. Therefore early osteoblast adhesion seems to be different on micro-rough and smooth titanium, but then hOB exhibited a similar proliferation rate. Our results show that surface roughness is not always increasing cell adhesion, and primary cells do require specific micro or nano-topography to spread and proliferate, unlike continuous cell lines which are easily growing on any substrate.

A second approach to control cell adhesion and spreading onto surfaces is the deposition of RGD sequence (Arg-Gly-Asp), the cell-binding domain shared by a number of bone related proteins, including collagen, fibronectin, bone sialoprotein, thrombospondin, vitronectin, etc. The process for immobilization of peptides on the surfaces is crucial, and the amount and pattern of immobilized peptide has to be controlled, as adhesion sites should have a specific spatial arrangement to be recognized by cell adhesion molecules. Inadequate distribution of such binding motifs has also been shown to promote apoptosis of cells, instead of enhanced adhesion.

In our lab polymers with irradiation treatment and RGD-addition were tested using human osteoblasts. In comparison with smooth surface, irradiated surfaces were found to promote cell adhesion and RGD immobilization was further increasing the number of cells highly spread, with well defined cytoskeleton, and evident stress fibers along the cell body. Therefore, RGD immobilization onto surfaces, if adequately tailored, is a powerful tool to recruit cells and to stimulate their function. Further improvements will make use of sequences which specifically bind osteoblasts to the functionalized surface.

3D scaffolds are conceived as bone substitutes for large bone defects: therefore they have to be able to host bone cells, to promote bone formation and to be replaced gradually by regenerated bone. They are mostly approved polymers which are modulated in terms of cristallinity, porosity, interconnections, etc. to get a controlled degradation rate, and often added with bone-like components (hydroxyapatite or b-tricalciumphosphate) to improve osteoconduction. Moreover, the scaffold can be loaded with cells or growth factors (BMPs), to fasten tissue regeneration, or with drugs for treatment of infection, cancer therapy, and so on. Naturally derived polymers, including the recent ‘bioscaffolds’, besides difficulty in preparation, suffer from poor control of enzymatic degradation and weak mechanical performance: therefore many research groups rely on synthetic polymers.

Poly-e-caprolactone (PCL) matrices, with micro- or macro-porosity, and with or without hydroxyapatite (HA) particles, have been extensively assayed in our Lab for their ability to support osteoblast growth and activity. In our hands the presence of HA particles within and onto the PCL scaffold was found to increase osteoblast adhesion and function. We have been able to detect surface colonization by continuous and primary bone cells, and also mineral formation after 3–4 weeks with proper additives, but the presence of viable cells in the ‘core’ of the scaffold is still a matter of debate. The employment of a spinner flask for cell seeding into matrices has been found to improve ‘conditioning’ of the scaffold, but not definitely cell entrance in depth. Confocal microscopy is to some extent faded by the autofluorescence of the polymer matrix, and light microscopy suffer from poor resolution. Results from our experience with hOB and MSC seeded on different 3D PCL scaffolds are presented.

Hydrophilic and hydrophobic polyurethane-based scaffolds (PU) were assayed in our lab, too. Despite high hydrophilicity and addition of the polymeric matrix with HA and b-tricalciumphosphate (TCP), hOB were not able to adhere and grow to confluence onto such porous polymers.

In summary, in vitro models with osteoblasts are a powerful tool to analyse biological compatibility of innovative surfaces or scaffolds, even if they are unable to model physiological function in vivo. Actually, that these models can work in the body has to be demonstrated in experimental in vivo testing, prior to clinical trial. However, the design and improvement of materials rely on the understanding of how cells basically respond to surfaces.

In conclusion, the challenge in bone engineering is to link clinical needs to material technology. In vitro and in vivo studies have demonstrated that the ability of materials to support bone formation can be enhanced by modifying the physical, chemical and biological characteristics of the surface, and surface micropatterning is a powerful tool for constructing elaborate intelligent bio-materials. But the biological response of bone cells and bone tissue, and therefore the orthopaedic research, is a critical step in material research and bone engineering.