Hydrophilic titanium surface‐induced macrophage modulation promotes pro‐osteogenic signalling

Objectives: As biomaterial‐induced modulation of mediators of the immune response may be a potential therapeutic approach to enhance wound healing events, the aim of this study was to delineate the effects of titanium surface modification on macrophage phenotype and function. Material and methods: Rodent bone marrow‐derived macrophages were polarized into M1 and M2 phenotypes and cultured on micro‐rough (SLA) and hydrophilic modified SLA (modSLA) titanium discs. Macrophage phenotype and cytokine secretion were subsequently assessed by immunostaining and ELISA, respectively. Osteoblast gene expression in response to culture in the M1 and M2 macrophage conditioned media was also evaluated over 7 days by RT‐PCR. Results: M1 macrophage culture on the modSLA surface promoted an M2‐like phenotype as demonstrated by marked CD163 protein expression, Arg1 gene expression and the secretion of cytokines that significantly upregulated in osteoblasts the expression of genes associated with the TGF‐ß/BMP signalling pathway and osteogenesis. In comparison, M2 macrophage culture on SLA surface promoted an inflammatory phenotype and cytokine profile that was not conducive for osteogenic gene expression. Conclusions: Macrophages are able to alter or switch their phenotype according to the signals received from the biomaterial surface. A hydrophilic micro‐rough titanium surface topography elicits a macrophage phenotype associated with reduced inflammation and enhanced pro‐osteogenic signalling

The effect of platelet proteins released in response to titanium implant surfaces on macrophage pro-inflammatory cytokine gene expression

Background and Purpose: Platelets are one of the earliest cell types to interact with surgically inserted titanium implants. This in vitro study investigated the effect of titanium surface-induced platelet releasate on macrophage cytokine gene expression

Titanium surface hydrophilicity modulates the human macrophage inflammatory cytokine response

Increased titanium surface hydrophilicity has been shown to accelerate dental implant osseointegration. Macrophages are important in the early inflammatory response to surgical implant placement and influence the subsequent healing response. This study investigated the modulatory effect of a hydrophilic titanium surface on the inflammatory cytokine expression profile in a human macrophage cell line (THP-1). Genes for 84 cytokines, chemokines, and their receptors were analyzed following exposure to (1) polished (SMO), (2) micro-rough sand blasted, acid etched (SLA), and (3) hydrophilic-modified SLA (modSLA) titanium surfaces for 1 and 3 days. By day 3, the SLA surface elicited a pro-inflammatory response compared to the SMO surface with statistically significant up-regulation of 16 genes [Tumor necrosis factor (TNF) Interleukin (IL)-1, Chemokine (C-C motif) ligand (CCL)-1, 2, 3, 4, 18, 19, and 20, Chemokine (C-X-C motif) ligand (CXCL)-1, 5, 8 and 12, Chemokine (C-C motif) receptor (CCR)-7, Lymphotoxin-beta (LTB), and Leukotriene B4 receptor (LTB4R)]. This effect was countered by the modSLA surface, which down-regulated the expression of 10 genes (TNF, IL-1 and , CCL-1, 3, 19 and 20, CXCL-1 and 8, and IL-1 receptor type 1), while two were up-regulated (osteopontin and CCR5) compared to the SLA surface. These cytokine gene expression changes were confirmed by decreased levels of corresponding protein secretion in response to modSLA compared to SLA. These results show that a hydrophilic titanium surface can modulate human macrophage pro-inflammatory cytokine gene expression and protein secretion. An attenuated pro-inflammatory response may be an important molecular mechanism for faster and/or improved wound healing. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 60-67, 2014

Titanium surface hydrophilicity enhances platelet activation

Titanium implant surface modification is a key strategy used to enhance osseointegration. Platelets are the first cells that interact with the implant surface whereupon they release a wide array of proteins that influence the subsequent healing process. This study therefore investigated the effect of titanium surface modification on the attachment and activation of human platelets. The surface characteristics of three titanium surfaces: smooth (SMO), micro-rough (SLA) and hydrophilic micro-rough (SLActive) and the subsequent attachment and activation of platelets following exposure to these surfaces were determined. The SLActive surface showed the presence of significant nanoscale topographical features. While attached platelets appeared to be morphologically similar, significantly fewer platelets attached to the SLActive surface compared to both the SMO and SLA surfaces. The SLActive surface however induced the release of the higher levels of chemokines beta-throm boglobulin and platelet factor 4 from platelets. This study shows that titanium surface topography and chemistry have a significant effect on platelet activation and chemokine release

The influence of titanium surface characteristics on macrophage phenotype polarization during osseous healing in type I diabetic rats: a pilot study

ObjectivesThis study assessed the effect of titanium surface modification on macrophage phenotype polarization and osseous healing under diabetic conditions

Effect of culture conditions and calcium phosphate coating on ectopic bone formation

This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaP-coated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients. We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation

The effects of implant topography on osseointegration under estrogen deficiency induced osteoporotic conditions: histomorphometric, transcriptional and ultrastructural analysis

Compromised bone quality and/or healing in osteoporosis are recognised risk factors for impaired dental implant osseointegration. This study examined the effects of (1) experimentally induced osteoporosis on titanium implant osseointegration and (2) the effect of modified implant surface topography on osseointegration under osteoporosis-like conditions. Machined and micro-roughened surface implants were placed into the maxillary first molar root socket of 64 ovariectomised and sham-operated Sprague-Dawley rats. Subsequent histological and SEM observations showed tissue maturation on the micro-rough surfaced implants in ovariectomised animals as early as 3 days post-implantation. The degree of osseointegration was also significantly higher around the micro-rough implants in ovariectomised animals after 14 days of healing although by day 28, similar levels of osseointegration were found for all test groups. The micro-rough implants significantly increased the early (day 3) gene expression of alkaline phosphatase, osteocalcin, receptor activator of nuclear factor kappa-B ligand and dentin matrix protein 1 in implant adherent cells. By day 7, the expression of inflammatory genes decreased while the expression of the osteogenic markers increased further although there were few statistically significant differences between the micro-rough and machined surfaces. Osteocyte morphology was also affected by estrogen deficiency with the size of the cells being reduced in trabecular bone. In conclusion, estrogen deficiency induced osteoporotic conditions negatively influenced the early osseointegration of machined implants while micro-rough implants compensated for these deleterious effects by enhancing osteogenic cell differentiation on the implant surface

Re-establishment of macrophage homeostasis by titanium surface modification in type II diabetes promotes osseous healing

Titanium surface mediated immunomodulation may address compromised post-implantation bone healing in\ua0diabetes mellitus. To assess\ua0in vitro\ua0phenotypic changes, M1 and M2 polarised Type 2 diabetic rat (Goto Kakizaki, GK) macrophages were cultured on micro-rough (SLA) or hydrophilic nanostructured SLA (modSLA) titanium. The\ua0in vivo\ua0effects of the SLA and modSLA surfaces on macrophage phenotype, wound-associated protein expression and bone formation were investigated using a critical-sized calvarial defect model. Compared to healthy macrophages, GK M2 macrophage function was compromised, secreting significantly lower levels of the anti-inflammatory cytokine IL-10. The modSLA surface attenuated the pro-inflammatory cellular environment, reducing pro-inflammatory cytokine production and promoting M2 macrophage phenotype differentiation. ModSLA also suppressed gene expression associated with macrophage multinucleation and giant cell formation and stimulated pro-osteogenic genes in co-cultured osteoblasts.\ua0In vivo, modSLA enhanced osteogenesis compared to SLA in GK rats. During early healing, proteomic analysis of both surface adherent and wound exudate material showed that modSLA promoted an immunomodulatory pro-reparative environment. The modSLA surface therefore successfully compensated for the compromised M2 macrophage function in Type 2 diabetes by attenuating the pro-inflammatory response and promoting M2 macrophage activity, thus restoring macrophage homeostasis and resulting in a cellular environment favourable for enhanced osseous healing

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts

Background: Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold's architecture on cellular gene expression however has not been fully elucidated. Methods: The design and fabrication of three different uniform pore structures (250, 500 and 750 μm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250-500-750 μm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. Results: The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. Conclusions: This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications

In vivo bone regeneration assessment of offset and gradient melt electrowritten (MEW) PCL scaffolds

Abstract Background Biomaterial-based bone tissue engineering represents a promising solution to overcome reduced residual bone volume. It has been previously demonstrated that gradient and offset architectures of three-dimensional melt electrowritten poly-caprolactone (PCL) scaffolds could successfully direct osteoblast cells differentiation toward an osteogenic lineage, resulting in mineralization. The aim of this study was therefore to evaluate the in vivo osteoconductive capacity of PCL scaffolds with these different architectures. Methods Five different calcium phosphate (CaP) coated melt electrowritten PCL pore sized scaffolds: 250 μm and 500 μm, 500 μm with 50% fibre offset (offset.50.50), tri layer gradient 250–500-750 μm (grad.250top) and 750–500-250 μm (grad.750top) were implanted into rodent critical-sized calvarial defects. Empty defects were used as a control. After 4 and 8 weeks of healing, the new bone was assessed by micro-computed tomography and immunohistochemistry. Results Significantly more newly formed bone was shown in the grad.250top scaffold 8 weeks post-implantation. Histological investigation also showed that soft tissue was replaced with newly formed bone and fully covered the grad.250top scaffold. While, the bone healing did not happen completely in the 250 μm, offset.50.50 scaffolds and blank calvaria defects following 8 weeks of implantation. Immunohistochemical analysis showed the expression of osteogenic markers was present in all scaffold groups at both time points. The mineralization marker Osteocalcin was detected with the highest intensity in the grad.250top and 500 μm scaffolds. Moreover, the expression of the endothelial markers showed that robust angiogenesis was involved in the repair process. Conclusions These results suggest that the gradient pore size structure provides superior conditions for bone regeneration