Early-stage macroporosity enhancement in calcium phosphate cements by inclusion of poly(N-vinylpyrrolidone) particles as a porogen

The incorporation of poly(DL-lactic-co-glycolic acid) (PLGA) particles into calcium phosphate cements (CPCs) is an effective strategy to enhance CPC macroporosity and degradation. However, bone regeneration is hindered until hydrolytic PLGA degradation starts a few weeks after implantation. Additionally, CPC and CPC/PLGA injectability and cohesion are suboptimal. In the current study, poly(N-vinylpyrrolidone) (PVP), a water-soluble polymer, was incorporated as a porogen in CPC and CPC/PLGA composites to enhance handling properties and early-stage macroporosity formation. Further, the effect of PVP molecular weight (Mw) and particle size was studied. The results showed that PVP incorporation increased both injectability and cohesion of the CPC pastes, especially with addition of high Mw PVP. Moreover, the in vitro degradation studies revealed that incorporation of PVP induced an initial mass loss during the first week of incubation. In combination with PLGA, small PVP particles induced a higher mass loss at an early stage than large PVP particles, but this effect was no longer apparent after 4 weeks of incubation. In contrast, the incorporation of low Mw PVP had a stronger effect on in vitro degradation in the long term compared to high Mw. Finally, the presence of PLGA porogens appeared to be necessary for adequate CPC degradation.Peer ReviewedPostprint (published version

Osteogenic differentiation driven by osteoclasts and macrophages

Introduction Osteoclasts are bone-resorbing cells closely related to bone turnover, whereas different macrophage subtypes contribute to bone fracture healing. As osteoclasts and macrophages share the same hematopoietic origin, the difference between both cell types on osteoblast coupling, crosstalk extent and consequent bone formation remains poorly understood. This study compares the potential of primary cells that are routinely considered as osteoclast and macrophage cultures on their ability to support osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Methods Human Peripheral Blood Mononuclear Cells (hPBMCs) were used to obtain macrophage or osteoclast cultures using appropriate stimulatory factors. With different seeding densities of hPBMCs, conditioned media from macrophage or osteoclast cultures were harvested for comparative evaluation of effects thereof on the osteogenic differentiation of hMSCs. Specific cytological staining was used to qualitatively evaluate macrophage and osteoclast cultures. Additionally, quantitative data on hMSC proliferation, osteogenic differentiation and mineralization were obtained via biochemical assays. Results Conditioned medium from osteoclast cultures obtained via low hPBMCs seeding densities, but not from high hPBMCs seeding densities or macrophages, stimulated hMSC osteogenic differentiation and mineralization. Upon cellular crosstalk, both pre-differentiated osteoclasts and non-polarized macrophages equally supported early hMSC osteogenic differentiation and mineralization, as confirmed by increased alkaline phosphatase levels within 7 days and increased calcium content within 14 days in comparison with undifferentiated controls. Initial hPBMCs seeding density strongly influences osteoclastogenesis and the paracrine effect of the resultant osteoclast population on the osteogenic differentiation of hMSCs. In addition, only in indirect coculture, macrophages provide similar stimulatory effects as pre-differentiated osteoclasts on the osteogenic differentiation of MSCs and mineralization. Conclusion Our results demonstrate stimulatory effects of osteoclast conditioned medium on hMSC osteogenic differentiation, depending on initial hPBMC seeding density. In addition, we show that osteoclast and macrophage cultures contain pools of polarized macrophages, which may be involved in the osteogenic effects. Our data provide insight into bone tissue engineering approaches by using multicellular interactions related to bone remodeling and healing for the in vitro modulation of osteogenic differentiation

Calcium phosphate cements: Optimization toward biodegradability

Synthetic calcium phosphate (CaP) ceramics represent the most widely used biomaterials for bone regenerative treatments due to their biological performance that is characterized by bioactivity and osteoconductive properties. From a clinical perspective, injectable CaP cements (CPCs) are highly appealing, as CPCs can be applied using minimally invasive surgery and can be molded to optimally fill irregular bone defects. Such CPCs are prepared from a powder and a liquid component, which upon mixing form a paste that can be injected into a bone defect and hardens in situ within an appropriate clinical time window. However, a major drawback of CPCs is their poor degradability. Ideally, CPCs should degrade at a suitable pace to allow for concomitant new bone to form. To overcome this shortcoming, control over CPC degradation has been explored using multiple approaches that introduce macroporosity within CPCs. This strategy enables faster degradation of CPC by increasing the surface area available to interact with the biological surroundings, leading to accelerated new bone formation. For a comprehensive overview of the path to degradable CPCs, this review presents the experimental procedures followed for their development with specific emphasis on (bio)material properties and biological performance in pre-clinical bone defect models.Peer ReviewedPostprint (published version

Pre-Clinical Evaluation of Biological Bone Substitute Materials for Application in Highly Loaded Skeletal Sites

The development of bone substitute materials (BSMs) intended for load-bearing bone defects is highly complicated, as biological and mechanical requirements are often contradictory. In recent years, biological BSMs have been developed which allow for a more efficient integration of the material with the surrounding osseous environment and, hence, a higher mechanical stability of the treated defect. However, while these materials are promising, they are still far from ideal. Consequently, extensive preclinical experimentation is still required. The current review provides a comprehensive overview of biomechanical considerations relevant for the design of biological BSMs. Further, the preclinical evaluation of biological BSMs intended for application in highly loaded skeletal sites is discussed. The selected animal models and implantation site should mimic the pathophysiology and biomechanical loading patterns of human bone as closely as possible. In general, sheep are among the most frequently selected animal models for the evaluation of biomaterials intended for highly loaded skeletal sites. Regarding the anatomical sites, segmental bone defects created in the limbs and spinal column are suggested as the most suitable. Furthermore, the outcome measurements used to assess biological BSMs for regeneration of defects in heavily loaded bone should be relevant and straightforward. The quantitative evaluation of bone defect healing through ex vivo biomechanical tests is a valuable addition to conventional in vivo tests, as it determines the functional efficacy of BSM-induced bone healing. Finally, we conclude that further standardization of preclinical studies is essential for reliable evaluation of biological BSMs in highly loaded skeletal sites

Effect of Osteoporosis on Well-Integrated Bone Implants

The installation of dental implants has become a common treatment for edentulous patients. However, concern exists about the influence of osteoporosis on the final implant success. This study evaluated whether an ovariectomy (OVX)-induced osteoporotic condition, induced eight weeks postimplantation in a rat femoral condyle, influences the bone response to already-integrated implants. The implants were inserted in the femoral condyle of 16 female Wistar rats. Eight weeks postimplantation, rats were randomly ovariectomized (OVX) or sham-operated (SHAM). Fourteen weeks later, animals were sacrificed, and implants were used for histological and histomorphometric analyses. A significant reduction in the quantity and quality of trabecular bone around dental implants existed in OVX rats in comparison to the SHAM group. For histomorphometric analysis, the bone area (BA%) showed a significant difference between OVX (34.2 ± 4.3) and SHAM (52.6 ± 12.7) groups (p < 0.05). Bone–implant contact (BIC%) revealed significantly lower values for all implants in OVX (42.5 ± 20.4) versus SHAM (59.0 ± 19.0) rats. Therefore, induction of an osteoporotic condition eight weeks postimplantation in a rat model negatively affects the amount of bone present in close vicinity to bone implants

Effect of Osteoporosis on Well-Integrated Bone Implants

The installation of dental implants has become a common treatment for edentulous patients. However, concern exists about the influence of osteoporosis on the final implant success. This study evaluated whether an ovariectomy (OVX)-induced osteoporotic condition, induced eight weeks postimplantation in a rat femoral condyle, influences the bone response to already-integrated implants. The implants were inserted in the femoral condyle of 16 female Wistar rats. Eight weeks postimplantation, rats were randomly ovariectomized (OVX) or sham-operated (SHAM). Fourteen weeks later, animals were sacrificed, and implants were used for histological and histomorphometric analyses. A significant reduction in the quantity and quality of trabecular bone around dental implants existed in OVX rats in comparison to the SHAM group. For histomorphometric analysis, the bone area (BA%) showed a significant difference between OVX (34.2 ± 4.3) and SHAM (52.6 ± 12.7) groups (p < 0.05). Bone–implant contact (BIC%) revealed significantly lower values for all implants in OVX (42.5 ± 20.4) versus SHAM (59.0 ± 19.0) rats. Therefore, induction of an osteoporotic condition eight weeks postimplantation in a rat model negatively affects the amount of bone present in close vicinity to bone implants

In vitro and in vivo effects of DNA-based coatings functionalized with vascular endothelial growth factor (VEGF)

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Composite Colloidal Gels Made of Bisphosphonate-Functionalized Gelatin and Bioactive Glass Particles for Regeneration of Osteoporotic Bone Defects

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Tunable calcium phosphate cement formulations for predictable local release of doxycycline

Background Osteomyelitis is a bacterial infection, which leads to bone loss. Local treatment focuses on elimination of bacteria, which is preferable for simultaneous management of the bone defect after sequestrectomy and bone reconstruction in one-stage treatment of osteomyelitis. Calcium phosphate cements (CPCs) have attracted increased attention as bone substitute material because of their injectability and in situ self-setting properties, which allow for minimally invasive surgical procedures and local drug delivery. Methods We herein established a system to achieve different release profiles of the antibiotic drug doxycycline from CPC by finetuning their formulation. These CPC formulations were generated via facile addition of hydrolytically degrading PLGA particles, varying doses of doxycycline, and addition of the lubricant CMC. Results The CPC formulations exhibited appropriate handling properties in terms of injectability and setting time. Furthermore, doxycycline release profiles showed an adequate burst release followed by a cumulative release of up to 100% over a period of 8 weeks. Importantly, the released doxycycline retained its antibacterial activity against Staphylococcus aureus, the major pathogen causing osteomyelitis. Using an in vivo implantation model, antibacterial efficacy was demonstrated by a rapid decrease of inoculated S. aureus at the CPC surface and within surrounding tissues. Conclusions Our data show the versatility of the CPC system toward local antibacterial therapy, extending its application beyond bone substitution.Peer ReviewedPostprint (published version