Johan van der Stok

Bone substitutes in the Netherlands - A systematic literature review

Autologous bone grafting is currently considered as the gold standard to restore bone defects. However, clinical benefit is not guaranteed and there is an associated 8-39% complication rate. This has resulted in the development of alternative (synthetic) bone substitutes. The aim of this systematic literature review was to provide a comprehensive overview of literature data of bone substitutes registered in the Netherlands for use in trauma and orthopedic surgery. Brand names of selected products were used as search terms in three available databases: Embase, PubMed and Cochrane. Manuscripts written in English, German or Dutch that reported on structural, biological or biomechanical properties of the pure product or on its use in trauma and orthopedic surgery were included. The primary search resulted in 475 manuscripts from PubMed, 653 from Embase and 10 from Cochrane. Of these, 218 met the final inclusion criteria. Of each product, structural, biological and biomechanical characteristics as well as their clinical indications in trauma and orthopedic surgery are provided. All included products possess osteoconductive properties but differ in resorption time and biomechanical properties. They have been used for a wide range of clinical applications; however, the overall level of clinical evidence is low. The requirements of an optimal bone substitute are related to the size and location of the defect. Calcium phosphate grafts have been used for most trauma and orthopedic surgery procedures. Calcium sulphates were mainly used to restore bone defects after tumour resection surgery but offer minimal structural support. Bioactive glass remains a potential alternative; however, its use has only been studied to a limited extent.

Selective laser melting-produced porous titanium scaffolds regenerate bone in critical size cortical bone defects.

Porous titanium scaffolds have good mechanical properties that make them an interesting bone substitute material for large bone defects. These scaffolds can be produced with selective laser melting, which has the advantage of tailoring the structures architecture. Reducing the strut size reduces the stiffness of the structure and may have a positive effect on bone formation. Two scaffolds with struts of 120‐µm (titanium‐120) or 230‐µm (titanium‐230) were studied in a load‐bearing critical femoral bone defect in rats. The defect was stabilized with an internal plate and treated with titanium‐120, titanium‐230, or left empty. In vivo micro‐CT scans at 4, 8, and 12 weeks showed more bone in the defects treated with scaffolds. Finally, 18.4 ± 7.1 mm3 (titanium‐120, p = 0.015) and 18.7 ± 8.0 mm3 (titanium‐230, p = 0.012) of bone was formed in those defects, significantly more than in the empty defects (5.8 ± 5.1 mm3). Bending tests on the excised femurs after 12 weeks showed that the fusion strength reached 62% (titanium‐120) and 45% (titanium‐230) of the intact contralateral femurs, but there was no significant difference between the two scaffolds. This study showed that in addition to adequate mechanical support, porous titanium scaffolds facilitate bone formation, which results in high mechanical integrity of the treated large bone defects.

Bone regeneration performance of surface-treated porous titanium

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.

Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats

The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

Additively manufactured porous tantalum implants

The medical device industrys interest in open porous, metallic biomaterials has increased in response to additive manufacturing techniques enabling the production of complex shapes that cannot be produced with conventional techniques. Tantalum is an important metal for medical devices because of its good biocompatibility. In this study selective laser melting technology was used for the first time to manufacture highly porous pure tantalum implants with fully interconnected open pores. The architecture of the porous structure in combination with the material properties of tantalum result in mechanical properties close to those of human bone and allow for bone ingrowth. The bone regeneration performance of the porous tantalum was evaluated in vivo using an orthotopic load-bearing bone defect model in the rat femur. After 12 weeks, substantial bone ingrowth, good quality of the regenerated bone and a strong, functional implant-bone interface connection were observed. Compared to identical porous Ti-6Al-4V structures, laser-melted tantalum shows excellent osteoconductive properties, has a higher normalized fatigue strength and allows for more plastic deformation due to its high ductility. It is therefore concluded that this is a first step towards a new generation of open porous tantalum implants manufactured using selective laser melting.

Predicting the therapeutic efficacy of MSC in bone tissue engineering using the molecular marker CADM1

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice.

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.

Inflammatory response and bone healing capacity of two porous calcium phosphate ceramics in critical size cortical bone defects

In the present study, two open porous calcium phosphate ceramics, β-tricalcium phosphate (β-TCP), and hydroxyapatite (HA) were compared in a critical-sized femoral defect in rats. Previous comparisons of these two ceramics showed significantly greater osteoinductive potential of β-TCP upon intramuscular implantation and a better performance in a spinal fusion model in dogs. Results of the current study also showed significantly more bone formation in defects grafted with β-TCP compared to HA; however, both the ceramics were not capable of increasing bone formation to such extend that it bridges the defect. Furthermore, a more pronounced degradation of β-TCP was observed as compared to HA. Progression of inflammation and initiation of new bone formation were assessed for both materials at multiple time points by histological and fluorochrome-based analyses. Until 12 days postimplantation, a strong inflammatory response in absence of new bone formation was observed in both ceramics, without obvious differences between the two materials. Four weeks postimplantation, signs of new bone formation were found in both β-TCP and HA. At 6 weeks, inflammation had subsided in both ceramics while bone deposition continued. In conclusion, the two ceramics differed in the amount of bone formed after 8 weeks of implantation, whereas no differences were found in the duration of the inflammatory phase after implantation or initiation of new bone formation.

Full-field strain measurement and fracture analysis of rat femora in compression test

There is a growing interest in studying the fracture behavior of bones, primarily due to the increasing societal burden of osteoporotic fractures. In addition, bone is one of the most important biological materials whose fracture behavior is not yet well understood. This is partly due to the fact that bone is a complex hierarchical material, and exhibits heterogeneous, anisotropic, and viscoelastic mechanical behavior. Understanding the fracture behavior of such a complex material requires application of a full-field strain measurement technique. Digital image correlation (DIC) is a relatively new full-field strain measurement technique that can be used for measurement of 3D surface strains during mechanical testing of different types of bones. In this study, we use the DIC technique to measure the surface strains during compression testing of two groups of rat femora. The first group of femora was harvested from young animals (12 weeks), while the second group was harvested from more mature animals (26 weeks). The surface strains are measured both in the linear range and close to the fracture. Using the measured data, we assess two strain-based fracture prediction criteria, namely equivalent strain fracture criterion and fracture limit diagram, to determine whether they can consistently predict the onset of fracture. The maximum load is measured to be 296±22 N (mean±SD) for young animals and 670±123 N for mature animals. It is shown that fracture in the vast majority of cases occurs in the area of maximum tensile strain. The equivalent strain fracture criterion predicts that the fracture occurs when the equivalent strain reaches 1.04±0.02% (average±SD) for young animals and 1.39±0.24% for mature animals. The fracture limit diagram predicts that the fracture occurs once the sum of major and minor principal surface strains reaches 0.63±0.23% for young animals and -0.63±0.30% for mature animals. Based on these numbers and consistency of the criteria with the strain values recorded at the fracture locations, it is concluded that the equivalent strain fracture criterion tends to be more consistent among the tested specimens.

Properties of commonly used calcium phosphate cements in trauma and orthopaedic surgery

Introduction Half of the population sustains at least one fracture during their lifetime, and the majority of these fractures heal successfully. Successful fracture healing requires the following five elements; (i) osteogenic cells (e.g., osteoblasts), (ii) osteoinductive stimuli (e.g., bone morphogenetic proteins); (iii) an osteoconductive matrix; (iv) adequate blood and nutrient supply, and (v) sufficient mechanical support. One or more elements can be compromised due to the existence of a bone defect. Bone defects are treated with bone grafts in order to avoid insufficient fracture healing. Insufficient fracture healing is encountered in 5–10% of the fractures, resulting in delayed union, malunion, or non-union.