Phosphoserine-modified calcium phosphate cements: bioresorption and substitution.

This work reports the effects of phosphoserine addition on the biodegradability of calcium phosphate cements. The characteristics of a phosphoserine-modified calcium phosphate cement without collagen in a large animal model are presented here for the first time. Critical size bone defects in the proximal tibia of 10 sheep were filled with the bone cement, and five sheep with empty defects were included as controls. The sheep were sacrificed after either 10 days or 12 weeks, and bones were processed for histological, histomorphometric and enzyme histochemical analyses as well as transmission electron microscopic examination. After 12 weeks, there was no significant reduction in either the implant or the bone defect cross-sectional area. Different amounts of fibrous tissue were observed around the implant and in the bone defect after 12 weeks. The direct bone-implant contact decreased after 12 weeks (p = 0.034). Although the implanted material properly filled the defect and promoted an initial activation of macrophages and osteoblasts, the resorption and simultaneous substitution did not reach expected levels during the experimental time course. Although other studies have shown that the addition of phosphoserine to calcium phosphate cements that have already been modified with collagen I resulted in an acceleration of cement resorption and bone regeneration, this study demonstrates that phosphoserine-modified calcium phosphate cements without collagen perform poorly in the treatment of bone defects. Efforts to use phosphoserine in the development of new composites should take into consideration the need to improve osteoconduction simultaneously via other means.

Modifications of a calcium phosphate cement with biomolecules--influence on nanostructure, material, and biological properties.

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding.

Glucuronic acid and phosphoserine act as mineralization mediators of collagen I based biomimetic substrates.

Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca(2+)/H( x )PO (4) ((3-x)) electrolyte at physiological pH and temperature. During mineralization of collagen-GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.

Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells.

Collagen and noncollagenous proteins of the extracellular bone matrix are able to stimulate bone cell activities and bone healing. The modification of calcium phosphate bone cements used as temporary bone replacement materials with these proteins seems to be a promising approach to accelerate new bone formation. In this study, we investigated adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells (hBMSC) on Biocement D/collagen composites which have been modified with osteocalcin and O-phospho-L-serine. Modification with osteocalcin was carried out by its addition to the cement precursor before setting as well as by functionalization of the cement samples after setting and sterilization. hBMSC were cultured on these samples for 28 days with and without osteogenic supplements. We found a positive impact especially of the phosphoserine-modifications but also of both osteocalcin-modifications on differentiation of hBMSC indicated by higher expression of the osteoblastic markers matrix metalloproteinase-13 and bone sialo protein II. For hBMSC cultured on phosphoserine-containing composites, an increased proliferation has been observed. However, in case of the osteocalcin-modified samples, only osteocalcin adsorbed after setting and sterilization of the cement samples was able to promote initial adhesion and proliferation of hBMSC. The addition of osteocalcin before setting results in a finer microstructure but the biological activity of osteocalcin might be impaired due to the sterilization process. Thus, our data indicate that the initial adhesion and proliferation of hBMSC is enhanced rather by the biological activity of osteocalcin than by the finer microstructure.

Bioactive silica-collagen composite xerogels modified by calcium phosphate phases with adjustable mechanical properties for bone replacement.

The development of composites has been recognized as a promising strategy to fulfil the complex requirements of biomaterials. The present study reports on the modification of a novel silica-collagen composite material by varying the inorganic/organic mass ratio and introducing calcium phosphate cement (CPC) as a third component. The sol-gel technique is used for processing, followed by xerogel formation under specific temperature and relative humidity conditions. Cylindrical monolithic samples up to 400mm(3) were obtained without any sintering processes. Various hierarchical phases of the organic component were applied, ranging from tropocollagen and collagen fibrils up to collagen fibers, each characterized by atomic force microscopy. Focusing on the application of fibrils, various inorganic/organic mass ratios were used: 100/0, 85/15 and 70/30; their influence on the structure of the composite material was demonstrated by scanning electron microscopy. The composition was extended by the addition of 25wt.% CPC which led to increased bioactivity by accelerating the formation of bone apatite layers in simulated body fluid. Synchrotron microcomputed tomography demonstrated the homogeneous distribution of the cement particles in the silica-collagen matrix. Compressive strength tests showed that the mechanical properties of the brittle pure silica gel are changed significantly due to collagen addition. The highest ultimate strength of about 115MPa at about 18% total strain was registered for the 70/30 silica-collagen composite xerogels. Incorporation of CPC lowered the gel's strength. By demonstrating differentiation of human monocytes into osteoclast-like cells, an important feature of the composite material regarding successful bone remodeling is fulfilled.

In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia.

The addition of chondroitin sulphate (CS) to bone cements with calcium phosphate has lead to an enhancement of bone remodeling and an increase in new bone formation in small animals. The goal of this study was to verify the effect of CS in bone cements in a large animal model simulating a clinically relevant situation of a segmental cortical defect of a critical size on bone-implant interaction and bone remodeling. The influence of adding CS to hydroxyapatite/collagen (HA/Col) composites on host response was assessed in a standard sheep tibia model. A midshaft defect of 3 cm was created in the tibiae of 14 adult female sheep. The defect was filled with a HA/Col cement cylinder in seven animals and with a CS-modified hydroxyapatite/collagen (HA/Col/CS) cement cylinder in seven animals. In all cases the tibia was stabilized with an interlocked universal tibial nail. The animals in each group were analyzed with X-rays, CT scans, histology, immunohistochemistry, and enzymehistochemistry, as well as histomorphometric measurements. The X-ray investigation showed a significantly earlier callus reaction around the HA/Col/CS implants compared to HA/Col alone. The amount of newly formed bone at the end point of the experiment was significantly larger around HA/Col/CS cylinders both in the CT scan and in the histomorphometric analysis. There were still TRAP-positive osteoclasts around the HA/Col implants after 3 months. The number of osteopontin-positive osteoblasts and the direct bone contact were significantly higher around HA/Col/CS implants. We conclude that addition of CS enhances bone remodeling and new bone formation around HA/Col composites.

O-phospho-L-serine: a modulator of bone healing in calcium-phosphate cements.

Bone substitution materials are seen as an alternative to autogenous bone transplants in the reconstruction of human bone structures. The aim of the present animal study was to evaluate the clinical handling and the conditions of bone healing after the application of a phosphoserine and collagen-I-modified calcium-phosphate cement (Biozement D). The application of phosphoserine is supposed to influence the texture of the extracellular matrix. Standardised bone defects were created in the lower jaw of 10 adult minipigs. These defects were reconstructed with a pasty calcium-phosphate cement mixture. After a healing time of 4 months, the animals were sacrificed. The mandibles of all animals were resected and non-decalcified histological sections of the areas of interest were prepared. The experiment was evaluated by means of qualitative histology and histomorphometry. The hydroxyapatite cement entirely hardened intraoperatively. Modelling and handling of the cement was facile and the margin fit to the host bone was excellent. Histology showed that resorption started in the periphery and proceeded exceptionally fast. The bony substitution, especially in phosphoserine-endowed cements, was very promising. After a healing period of 4 months, phosphoserine cements showed a bone regeneration of nearly two-thirds of the defect sizes. In the applied animal experiment, the newly developed hydroxyapatite collagen-I cement is well suited for bone substitution due to its easy handling, its excellent integration and good resorption characteristics. The addition of phosphoserine is very promising in terms of influencing resorption features and bone regeneration.

Histologic study of incorporation and resorption of a bone cement-collagen composite: an in vivo study in the minipig.

OBJECTIVE: Calcium phosphates are clinically established as bone defect fillers. They have the capability of osseoconduction and are characterized by a slow resorption process. The present study evaluated the suitability of a newly developed calcium phosphate cement modified with collagen type I. STUDY DESIGN: The modified cement paste was inserted in differently designed defects of 10 minipigs. Further, an alveolar ridge augmentation was performed, applying the cement paste. The cement hardened in situ during the operation, forming a hydroxyapatite collagen composite. Animals were sacrificed after 1, 3, 6, 12, and 18 months. The tissue integration and resorption process was then evaluated using nondecalcified microsections. All animals were evaluated for histology. RESULTS: The implanted material showed osseoconductive characteristics. Resorption started from the edge of the defect zone, and bone substitution followed rapidly. Twelve months after placement of the cement, complete remodeling was observed. CONCLUSION: It can be concluded that the applied hydroxyapatite-collagen cement composite shows good resorption and bone integration.

Effect of chondroitin sulphate on material properties and bone remodelling around hydroxyapatite/collagen composites.

Chondroitin sulphate (CS) has an anti-inflammatory effect and increases the regeneration ability of injured bone. The goal of this study was to characterize the material properties and osteoconductive potency of calcium phosphate bone cements modified with CS. The early interface reaction of cancellous bone to a nanokristalline hydroxyapatite cement containing type I collagen (HA/Coll) without and with CS (HA/Coll/CS) in a rat tibia model was evaluated. Cylindrical implants were inserted press-fit into defect of the tibial head. Six specimens per group were analyzed at 2, 4, 7, 14, and 28 days. HA/Coll/CS composite cylinders showed a 15% increase in compressive strength and by investigations with powder X-ray diffraction more nontransformed cement precursor was found. The microstructures of both types of implants were similar. A significantly higher average number of TRAP positive osteoclasts and ED1 positive mononuclear cells were observed in the interface around HA/Coll/CS implants on day 4 and 7 (p < 0.05). At 28 days the direct bone contact and the percentage of newly formed bone were significantly higher around HA/Coll/CS implants (p < 0.05). The addition of CS appears to enhance bone remodelling and new bone formation around HA/Coll composites in the early stages of bone healing. Possible mechanisms are discussed.

Effect of modification of hydroxyapatite/collagen composites with sodium citrate, phosphoserine, phosphoserine/RGD-peptide and calcium carbonate on bone remodelling.

This study describes the early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite cement containing type I collagen (HA/Coll) and its modifications with sodium citrate (CI), calcium carbonate (CA), phosphoserine (P) and phosphoserine plus RGD-peptide (RGD). Cylindrical implants of HA/Coll and its modifications were inserted into the tibia of Wistar rats. We analysed 6 specimens per group at days 2, 4, 7, 14 and 28. CI, P and RGD modifications showed improved material properties (finer microstructure and higher compressive strength) compared to CA and HA/Coll implants. The powder X-ray diffraction (XRD) showed that the addition of P and CI led to an increase of alpha-TCP peaks while the diffraction patterns of the non-modified cement (HA/Coll) were quite similar with that of natural bone. All of the implants healed without adverse reactions. A significantly higher number of TRAP-positive osteoclasts were observed around CI, RGD and P on day 7 compared to CA and HA/Coll. Around CI, P and RGD a significantly delayed increase of ED1-positive mononuclear cells was detected. The amount of direct bone contact after 28 days was significantly higher around CI, P and RGD compared to CA and HA/Coll implants. The addition of CI, P and RGD appears to enhance bone remodelling at the early stages of bone healing, leading to increased bone formation around HA/Coll composite cements.

In vitro ossification and remodeling of mineralized collagen I scaffolds.

A promising strategy of bone tissue engineering is to repair bone defects by implanting biodegradable scaffolds that can undergo remodeling and be replaced completely by autologous bone tissue. For this purpose, it is necessary to create scaffolds that can be degraded by osteoclasts and enable osteoblasts to build new mineralized bone matrix. In order to achieve this goal a new porous material has been developed using biomimetically mineralized collagen I. These scaffolds were co-cultured with osteoclast-like cells and osteoblasts in order to characterize the capacity of these cells to remodel the material in vitro. It was possible to show the development of biologically active osteoclast- like cells that were able to invade and degrade the scaffold. They degraded the scaffold by internalizing it as intracellular vesicles, thereby making room for osteoblasts to invade and build new bone matrix. In addition, it could be shown that osteoblasts proliferated, differentiated, and produced new mineralized extracellular matrix. Hence, it could be shown that co-culture of osteoclastlike cells and osteoblasts on biomimetically mineralized collagen I is a promising approach for bone tissue engineering. In addition, it can be applied to study the process of bone remodeling in vitro.

Osteocalcin enhances bone remodeling around hydroxyapatite/collagen composites.

The effect of osteocalcin (OC), an extracellular bone matrix protein, on bone healing around hydroxyapatite/collagen composites was investigated. Cylindrical nanocrystalline hydroxyapatite implants of 2.5-mm diameter containing 2.5% biomimetically mineralized collagen type I were inserted press-fit into the tibial head of adult Wistar rats. To one implant group, 10 mug/g OC was added. Six specimens per group were analyzed at 2, 7, 14, 28, and 56 days. After 14 days, newly formed woven bone had reached the implant surface of the OC implants whereas a broad fibrous interface could still be observed around controls. Woven bone was formed directly around both implant groups after 28 days and had been replaced partially by lamellar bone around the OC implants only. No significant differences in total bone contact were seen between both groups after 56 days. The higher number of phagocytosing cells and osteoclasts characterized immunohistochemically with ED1, cathepsin D, and tartate-resistant alkaline phosphatase around the OC implants at the early stages of bone healing suggests an earlier onset of bone remodeling. The earlier and increased expression of bone-specific matrix proteins and multifunctional adhesion proteins (osteopontin, bone sialoprotein, CD44) at the interface around the OC implants indicates that OC may accelerate bone formation and regeneration. This study supports the observations from in vitro studies that OC activates both osteoclasts and osteoblasts during early bone formation.