Gert J. Meijer

Cell-Based Bone Tissue Engineering

The authors review the available data on bone tissue engineering and discuss possible new research areas that could help to make bone tissue engineering a clinical success.

Cell based bone tissue engineering in jaw defects.

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. At the end of the procedure, this viable bone substitute was not only re-implanted in the patient, but also simultaneously subcutaneously implanted in mice to prove its osteogenic potency. In all patients, a viable bone substitute was successfully constructed, which was proven by bone formation after subcutaneous implantation in mice (ectopic bone formation). However, the same construct was reluctant to form bone in patients with intra-oral osseous defects (orthotopic bone formation). Although biopsies, taken 4 months after reconstructing the intra-oral bone defect, showed bone formation in 3 patients, only in 1 patient bone formation was induced by the tissue-engineered construct. Although bone tissue engineering has proven its value in animal studies, extra effort is needed to make it a predictable method for reconstruction jaw defects in humans. To judge its benefit, it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteogenesis).

Influence of surgical technique and surface roughness on the primary stability of an implant in artificial bone with different cortical thickness: a laboratory study

OBJECTIVE The aim of this biomechanical study was to assess the interrelated effect of both surface roughness and surgical technique on the primary stability of dental implants. MATERIAL AND METHODS For the experiment, 160 screw-designed implants (Biocomp), with either a machined or an etched surface topography, were inserted into polyurethane foam blocks (Sawbones). As an equivalent of trabecular bone, a density of 0.48 g/cm(3) was chosen. To mimic the cortical layer, on top of these blocks short-fibre-filled epoxy sheets were attached with a thickness varying from 0 to 2.5 mm. The implant sites were prepared using either a press-fit or an undersized technique. To measure the primary stability of the implant, both the insertion and the removal torques were scored. RESULTS Independent of the surgical technique used, both implant types showed an increased insertion and removal torque values with increasing cortical thickness, although >2 mm cortical layer no further increase in insertion torque was observed. In the models with only trabecular bone (without cortical layer) and with a 1 mm cortical layer, both implant types showed a statistically higher insertion and removal torque values for undersized compared with the press-fit technique. In addition, etched implants showed a statistically higher insertion and removal torque mean values compared with machined implants. In the models with 2 and 2.5 mm cortical layers, with respect to the insertion torque values, no effect of either implantation technique or implant surface topography could be observed. CONCLUSION The placement of etched implants in synthetic bone models using an undersized preparation technique resulted in enhanced primary implant stability. A correlation was found between the primary stability and the cortical thickness. However, at or above a cortical thickness of 2 mm, the effect of both an undersized surgical approach, as also the presence of a roughened (etched) implant surface, had no extra effect. Besides the mechanical aspects, the biological effect of undersized drilling, i.e. the bone response on the extra insertion torque forces should also be elucidated. Therefore, additional in vivo studies are needed.

Influence of the surgical technique and surface roughness on the primary stability of an implant in artificial bone with a density equivalent to maxillary bone: a laboratory study.

OBJECTIVE The aim of this biomechanical study was to assess the effect of surgical technique and surface roughness on primary implant stability in low-density bone. MATERIAL AND METHODS Eighty screw-shaped (Biocomp) implants with machined or etched surface topography were inserted into a low-density bone equivalent. Solid rigid polyurethane blocks (Sawbones) with two different bone densities (group A=0.32 g/cm(3); group B=0.48 g/cm(3)) were used that are very similar to the density of the maxilla. The implant sites were prepared either by a press-fit or by an undersized technique. Peak insertion and removal torques were measured using a Digital torque gauge instrument. RESULTS Independent of the surgical technique used, both implant types showed an increased mean insertion and removal torque value with increasing bone density. Insertion and removal torque values were 54.3+/-5.3 and 43.5+/-6.5 N cm for group A and 89.3+/-7.6 and 55+/-9.1 for group B, respectively. For group A and B, both implant types showed statistically higher insertion and removal torque mean values for the undersized compared with the press-fit technique (P<0.01). In addition, etched implants showed statistically higher insertion and removal torque mean values compared with machined implants (P<0.01). CONCLUSION The placement of etched implants in synthetic bone models using an undersized preparation technique resulted in enhanced primary implant stability; further, a correlation was found between primary stability and bone density of the synthetic bone, implying that in case of an implant site with low bone density, by changing the surgical technique and choosing an implant with an optimal surface roughness, the primary stability can be enhanced significantly.

Concise Review: Cell-Based Strategies in Bone Tissue Engineering and Regenerative Medicine

Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell‐based constructs. So far, preclinical studies have demonstrated that cell‐based tissue‐engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell‐based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation.

The Effect of Platelet-Rich Plasma In Vitro on Primary Cells: Rat Osteoblast-like Cells and Human Endothelial Cells

The aim of this study was to evaluate the effects of standardized platelet-rich plasma (PRP) concentrates from 10 human donors on cellular behavior. The standardized PRPs used were fivefold average and fivefold maximum baseline values in whole blood. Both these standardized PRPs were characterized by determining platelet numbers and subsequently growth factor concentrations in activated PRPs, called PRP derivatives. Platelet numbers in both types of standardized PRPs were significantly increased compared with whole blood. Further, both PRP derivatives contained significantly higher concentrations of platelet-derived growth factor-AA, platelet-derived growth factor-AB, and transforming growth factor-beta 1. Vascular endothelial growth factor concentrations were significantly elevated in only the most concentrated PRP derivative. Cell culture experiments with osteoblast-like cells showed that both PRP derivatives stimulated cell proliferation without inducing cell differentiation, whereas tube formation in endothelial cell cultures was significantly increased by adding low volume percentages of PRP derivative (2%–8%). Consequently, it can be concluded that there is no direct relationship between the number of platelets and the level of growth factors released from these platelets. PRP derivatives have the potency to stimulate angiogenesis dose dependently, while lacking the capacity to induce osteogenic differentiation. Yet, the proliferation of osteoblast-like cells can significantly be enhanced by supplementation of PRP derivatives.

The quantitative assessment of peri-implant bone responses using histomorphometry and micro-computed tomography.

In the present study, the effects of implant design and surface properties on peri-implant bone response were evaluated with both conventional histomorphometry and micro-computed tomography (micro-CT), using two geometrically different dental implants (Screw type, St; Push-in, Pi) either or not surface-modified (non-coated, CaP-coated, or CaP-coated+TGF-beta1). After 12 weeks of implantation in a goat femoral condyle model, peri-implant bone response was evaluated in three different zones (inner: 0-500 microm; middle: 500-1000 microm; and outer: 1000-1500 microm) around the implant. Results indicated superiority of conventional histomorphometry over micro-CT, as the latter is hampered by deficits in the discrimination at the implant/tissue interface. Beyond this interface, both analysis techniques can be regarded as complementary. Histomorphometrical analysis showed an overall higher bone volume around St compared to Pi implants, but no effects of surface modification were observed. St implants showed lowest bone volumes in the outer zone, whereas inner zones were lowest for Pi implants. These results implicate that for Pi implants bone formation started from two different directions (contact- and distance osteogenesis). For St implants it was concluded that undersized implantation technique and loosening of bone fragments compress the zones for contact and distant osteogenesis, thereby improving bone volume at the interface significantly.

In vivo bone response and mechanical evaluation of electrosprayed CaP nanoparticle coatings using the iliac crest of goats as an implantation model

Recent trends in clinical implantology include the use of endosseous dental implant surfaces embellished with nano-sized modifications. The current study was initiated to evaluate the mechanical properties, as well as the potential beneficial effects, of electrosprayed CaP nanoparticle-coated (nano-CaP) implants on the in vivo osteogenic response, compared with grit-blasted, acid-etched (GAE) implant surfaces as controls. For this purpose nano-CaP coatings were deposited on cylindrical screw-type (St) implants and implanted bilaterally into the iliac crest of goats for 6weeks. In addition to histological and histomorphometrical analyses, insertion torque and removal torque values were measured on implant placement and retrieval, respectively. The present study showed similar insertion and removal torque values for nano-CaP-coated and GAE control implants, with no statistically significant increase in torque value during the implant period for either group. With regard to bone-implant contact and peri-implant bone volume, no significant differences were found between nano-CaP-coated and GAE implants after 6weeks implantation. In conclusion, this study has demonstrated that in situations in which implants are placed in a non-compromised situation using a standard press fit implantation strategy the performance of electrosprayed nano-CaP coatings is comparable with GAE implants, both with respect to implant fixation and bone healing response.

The effect of alkaline phosphatase coated onto titanium alloys on bone responses in rats.

The enzyme alkaline phosphatase (ALP) was recently proposed as an implant coating material in order to improve the biological performance of orthopedic and dental implants. The present study evaluated the in vivo bone response to electrosprayed coatings, consisting of ALP, calcium phosphate (CaP) or a combination thereof (composite coating: ALP+CaP) compared to non-coated controls (gritblasted and acid etched). A total of 80 implants (n=10) with a gap of 1.0mm, was implanted intramedullary and bilaterally into the femurs of 80 rats. After 1 and 4 weeks, bone response was evaluated qualitatively (histology) and quantitatively (histomorphometry). The results of this study show that all electrosprayed coatings (ALP, CaP, ALP+CaP) significantly improve osteoconduction compared to non-coated controls after 4 weeks of implantation, without significant differences among these coated groups. Consequently, the results indicate that ALP-coatings improve the osteogenic response to a comparable extent as CaP-coatings or an ALP+CaP composite coating. In conclusion, the current study proofs that ALP-coatings have potential as bone implant coatings, though long-term data remain to be obtained. From a clinical perspective, it was observed that the process of osteoconduction is related to positional determinants, which needs to be taken into account when analyzing data on bone response.

Periodontal regeneration using an injectable bone cement combined with BMP‐2 or FGF‐2

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein‐2 (BMP‐2) or fibroblast growth factor‐2 (FGF‐2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP‐2 and CaP + FGF‐2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP‐2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP‐2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF‐2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP‐2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP +  FGF‐2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP‐2 group. The combination of a topical application of FGF‐2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration. Copyright