Demineralized Bone Particles Research Articles

Characterization and Potential of a Bilayered Hydrogel of Gellan Gum and Demineralized Bone Particles for Osteochondral Tissue Engineering.

Osteochondral (OC) tissue engineering (TE) is a promising strategy to regenerate acute or degenerative chondral and OC lesions. However, advancing a proper model for OC TE is still under way. Herein, a bilayer hydrogel (BH) based on gellan gum (GG) hydrogel and demineralized bone particles (DBPs) is suggested as a new model. The BH composite can be fabricated easily with a cell-friendly biomaterial and cross-linker. The BH composite was characterized by a morphological method and physicochemical aspect. The mechanical and rheological characters were further confirmed to verify its applicability in OC TE. The thermodynamic property of the composite was determined to analyze thermal stability and interaction among matrices. The bioactivity of the material was studied by treating simulated body fluid (SBF) solution for 28 days to examine the formation of crystalline structure in the BH construct. In vitro studies were carried out to study the viability and biochemical characters of the developed biomaterial. An in vivo study was performed to analyze the biocompatibility of the material and regeneration of the injured OC region implanted with BH composites. The data displayed stable physicochemical properties and mechanical characters when the DBPs were incorporated with a proper amount. The bioactivity of the DBP-loaded hydrogels displayed a high amount of apatite formation. The cytotoxicity of the fabricated material was low, which allows application in vitro and in vivo. The biochemical studies displayed a high level of alkaline phosphatase (ALP) activity and gene expression, which shows promising application of DBP-loaded GG in the bone layer of the BH model. The long-term in vivo study displayed excellent biocompatibility and great potential in the OC defected region. Overall, these results suggest the significance of combined and innovative approaches to improve the therapeutic strategies for OC regeneration, and the BH model suggested in this study can be a promising biomaterial model for OC TE.

In-vivo Performance of Seven Commercially Available Demineralized Bone Matrix Fiber and Putty Products in a Rat Posterolateral Fusion Model.

Introduction: Demineralized bone matrix (DBM) is a widely used bone graft in spinal fusion. Most commercial DBMs are composed of demineralized bone particles (~125–800 microns) suspended in a carrier that provides improved handling but dilutes the osteoinductive component. DBM fibers (DBF) provide improved osteoconductivity and do not require a carrier. It has been suggested that 100% DBF may offer improved performance over particulate-based DBMs with carrier.Study Design: Seven commercially available DBM products were tested in an athymic rat posterolateral fusion model. There were four 100% DBFs, two DBFs containing a carrier, and one particulate-based DBM containing carrier.Objective: The study objectives were to evaluate the in vivo performance: (1) compare fusion rate and fusion maturity of six commercially available DBFs and one particulate-based DBM, and (2) assess the effect of carrier on fusion outcomes for DBFs in a posterolateral fusion model.Methods: The DBF/DBM products evaluated were: StrandTM Family, Propel® DBM Fibers, Vesuvius® Demineralized Fibers, Optium® DBM Putty, Grafton® DBF, Grafton Flex, and DBX® Putty. Single-level posterolateral fusion was performed in 69 athymic rats. Fusion was assessed bilaterally after 4 weeks by manual palpation, radiograph and CT for bridging bone. Fusion mass maturity was assessed with a CT maturity grading scale and by histology. Statistical analysis was performed using Fishers Exact Test for categorical data and Kruskal-Wallis Test for non-parametric data.Results: Strand Family achieved 100% fusion (18/18) by manual palpation, radiographic and CT evaluation, significantly higher than Propel Fibers, Vesuvius Fibers, Optium Putty, and DBX Putty, and not statistically higher than Grafton DBF and Grafton Flex. Strand Family provided the highest fusion maturity, with CT maturity grade of 2.3/3.0 and 89% mature fusion rate. Fusion results suggest a detrimental effect of carrier on fusion performance.Conclusions: There were large variations in fusion performance for seven commercially available DBM products in an established preclinical fusion model. There were even significant differences between different 100% DBF products, suggesting that composition alone does not guarantee in vivo performance. In the absence of definitive clinical evidence, surgeons should carefully consider available data in valid animal models when selecting demineralized allograft options.

Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

Osteogenic Differentiation of Rat Adipose Stem Cells in Demineralized Bone Particles Sponges

Osteogenic Differentiation of Rat Adipose Stem Cells in Demineralized Bone Particles Sponges

Evaluation of Osteogenesis on Duck`s Feet Derived Collagen and Demineralized Bone Particles Sponges

본 연구에서는 오리발 콜라겐(duck’s feet derived collagen, DC)과 탈미네랄화된 골분(demineralized bone particles, DBP)을 3:1, 1:1, 1:3 비율로 혼합하여 스펀지를 제조하였다. 제조한 스펀지의 특성분석을 위해 압축강도, 다공성, SEM 등을 측정하였으며, 골수 유래 줄기세포(BMSC)를 비율별 DC/DBP 스펀지에 파종하여, 세포 부착, 증식 및 골 분화를 확인하고자, SEM, MTT, ALP, RT-PCR 및 조직학적 염색을 통한 분석을 실시하였다. 그 결과, 1:1 DC/DBP 스펀지에서 골 분화 효과가 우수한 것을 확인하였으며, 이를 골 재생을 위한 생체재료로 활용할 수 있을 것으로 기대된다.

A study of a three-dimensional PLGA sponge containing natural polymers co-cultured with endothelial and mesenchymal stem cells as a tissue engineering scaffold

The interaction between vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in a complex hemodynamic and mechanical environment plays an important role in the control of blood vessel growth and function. Despite the importance of VSMCs, substitutes are needed for vascular therapies. A potential VSMC substitute is human adult bone marrow derived mesenchymal stem cells (hMSCs). In this study, the effect of poly(lactic-co-glycolic acid) (PLGA) scaffolds containing three natural polymers (demineralized bone particles, silk, and small intestine submucosa) on the phenotype of MSCs and SMCs cultured with or without ECs was investigated. The study objective was to create a media equivalent for a tissue engineered blood vessel using PLGA, natural polymers, and MSCs co-cultured with ECs. The PLGA containing the natural polymers silk and SIS showed increased proliferation and cell adhesion. The presence of silk and DBP promoted a MSC phenotype change into a SMC-like phenotype at the mRNA level; however these differences at the protein level were not seen. Additionally, PLGA containing SIS did not induce SMC gene or protein upregulation. Finally, the effect of ECs in combination with the natural polymers was tested. When co-cultured with ECs, the mRNA of SMC specific markers in MSCs and SMCs were increased when compared to SMCs or MSCs alone. However, MSCs, when co-cultured with ECs on PLGA containing silk, exhibited significantly increased α-SMA and calponin expression when compared to PLGA only scaffolds. These results indicate that the natural polymer silk in combination with the co-culture of endothelial cells was most effective at increasing cell viability and inducing a SMC-like phenotype at the mRNA and protein level in MSCs.

탈미네랄골분이 첨가된 알지네이트 스펀지에서 조직공학적 연골 재건

알지네이트와 탈미네랄골분(DBP) 하이브리드 스펀지는 각각 DBP가 0, 10, 20, 40 및 80% 첨가된 스펀지로, 연골 재건을 위해 제작되었다. 세포의 증식은 MTT를 통해 관찰했으며, 형태학적, 조직학적, 생물학적 관찰 그리고 RT-PCR을 1, 2, 및 3 주에 실시하였다. 세포생존율에 있어서 20% DBP/알지네이트 스펀지에서 다른 스펀지보다도 가장 좋은 결과를 보였으며, 광학전자현미경 관찰에서 또한 20% 스펀지에서 가장 좋은 세포부착률과 증식률을 확인하였다. 마지막으로, 조직학적 평가에서 20% 스펀지에서 연골세포의 표현형이 가장 잘 유지됨과 동시에 산성뮤코 다당류와 제2형 콜라겐이 잘 형성된 것을 확인하였다. 이로써 DBP/알지네이트 하이브리드 스펀지는 연골 조직공학을 위한 지지체로써 활용될 수 있을 것이다. Demineralized bone particles (DBP) and alginate hybrid sponges were fabricated at 10, 20, 40 and 80% DBP/alginate hybrid ratios for seeding chondrocyte. Cell proliferation was measured via MTT assay. Morphological observation, histology, biological assay and RT-PCR were performed at each time point 1, 2 and 3 weeks. The cell viability was better in 20% DBP/alginate sponges than in other sponges. SEM results showed that more attached and more proliferated cells in the 20% DBP/alginate sponges with the lapse of time. Finally, histochemical assay results showed that the phenotype of chondrocyte was well maintained and both acidic mucopolysaccharide and type II collagen was well formed at 20% sponges. This study suggested that DBP/alginate sponge may serve as a potential cell delivery vehicle and a structural basis for tissue engineered articular cartilage.

Postraumatic frontal sinus obliteration with calvarial bone dust and demineralized bone matrix: a long term prospective study and literature review

Defining the ideal material for frontal sinus obliteration remains controversial. Autogenous cancellous bone is effective because of its biological properties: it undergoes fast revascularization acting as an active scaffold for bone healing, but is linked to additional donor site morbidity. Bone dust harvesting from the skull surface produces no sequelae but availability is limited. Many efforts have been made to overcome these drawbacks, and an ideal bone substitute sought. Demineralized Bone Matrix (DBX; Musculoskeletal Transplant Foundation, Edison, NJ, USA) is a commercially available product composed of demineralized bone particles reduced after proper processing of human bone in combination with sodium hyaluronate. It generates an osteoconductive surface and it is also a source of osteoinductive factors. Radiological follow-up using computed tomography is a very reliable method of following-up ossification and detecting the early signs of possible complications. The authors present their clinical series of postraumatic frontal sinus obliteration using a mixture of calvarial bone dust and DBX shell, with long-term radiological monitoring. The technique was demonstrated to be effective, reliable, stable in the long term and associated with minimal morbidity.

Effect of demineralized bone particles (DBP) on cell growth and ECM secretion in PLGA/DBP hybrid scaffold for cartilage tissue engineering

In this study, we investigated the effect of deminerized bone particles (DBP) on the regeneration of chondrocyte in poly(lactic-co-glycolic acid) (PLGA)/DBP hybrid scaffolds. PLGA/DBP hybrid scaffolds were designed by varying the DBP content with 0, 10, 20, 40, and 80 w/t% and manufactured by a solvent casting/saltleaching method. We evaluated the potential of PLGA/DBP hybrid scaffolds for cartilage regeneration. Chondrocytes of New Zealand white rabbits were seeded on PLGA/DBP hybrid scaffolds. MTT assay, glycosaminoglycan (GAG) assay, Hematoxylin-Eosin (H&E) staining, Safrainin-O staining, and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the effect of the DBP on cell proliferation and extracellular matrix (ECM) production. As the DBP content of these scaffolds became higher, the rate of cell growth and the amount of GAG increased. The PLGA/DBP hybrid scaffolds with DBP content showed better cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation, and sGAG production. In conclusion, PLGA/DBP hybrid scaffolds promote the potential cell delivery vehicle and act as the structural basis for cartilage tissue engineering. Open image in new window

Rapid and consistent porcine model for chronic total occlusions in the peripheral vasculature

Development and testing of treatment options for chronic total occlusions (CTO) still remains one of the last frontiers of vascular interventions. The lack of a consistent CTO animal model is one of the hurdles precluding efficient treatment advances in this field.We describe here a porcine model of a calcified CTO achieved by an intravascular delivery of de‐mineralized bone matrix (DBM) products in the femoral arteries. DBM is essentially de‐mineralized allograft bone particles with osteoinductive properties. In this study, the DBM material was delivered to each vessel via a guide catheter based approach. At 28 days, each treated vessel was evaluated angiographically, the animals were sacrificed and the treated vessels segments were assessed histopathologically. CTO was detected in 73% of the treated vessels. Morphologically, the CTO lesions were characterized by a mixed tissue growth composed of well vascularized collagenous matrix, mineralized and un‐mineralized bone fragments and variable mononuclear inflammation. This tissue reaction appeared well adhered to the vascular tunica media. Vascular channels lined by endothelium were present in vessels with incomplete chronic occlusion.This animal model can provide a platform for 1) development and testing of new CTO crossing devices, 2) evaluation of stents in calcified vessels 3) identification and evaluation of pharmacological entities in context of calcification and 4) development and evaluation of decalcification treatments.

Mechanisms of Osteoinduction/Chondroinduction by Demineralized Bone

Demineralized bone is widely used for osseous reconstructive procedures. Bone morphogenetic proteins (BMPs) have been studied as the putative active components of demineralized bone; recombinant BMPs are Food and Drug Administration-approved for use with scaffold carriers. An experimental three-dimensional in vitro model was developed to allow comparison of early changes in signaling and gene expression in cells exposed to demineralized bone particles (DBPs) or to BMP and/or transforming growth factor beta (TGF-beta). The culture device consists of a bilayer of porous collagen lattice that contains either a packet of DBPs or an insert of the dense collagen scaffold with BMP and/or TGF-beta. As expected, BMP and TGF-beta induced distinct signaling pathways and events in human dermal fibroblasts; DBPs signaled both pathways and their target genes. A mixture of BMP and TGF-beta stimulated the production of extracellular matrix chondroitin that approximated the composition that was stimulated by DBPs, but to a lesser amount. Although BMP was originally isolated as the active factor in DBPs, it does not show DBPs' effects on signaling and chondroinduction in vitro. Multiple members of the TGF-beta superfamily and other constituent factors may be involved in skeletal induction of human dermal fibroblasts by DBPs. This in vitro culture system may be useful to provide a rational basis for designing improved osteoinductive/chondroinductive materials.

Reduction of Inflammatory Reaction of Poly(D,L-Lactic-Co-Glycolic Acid) Using Demineralized Bone Particles

Poly(lactide-co-glycolic acid) (PLGA) has been widely applied to tissue engineering as a good biocompatible material because of its biodegradability and nontoxic metabolites, but how the inflammatory reaction of PLGA on the surrounding tissue in vivo is reduced has not been discussed sufficiently. We hypothesized that the cells neighboring the PLGA implant might have an inflammatory response that could be reduced by impregnating demineralized bone particles (DBPs) into the PLGA. We manufactured five different ratios of DBP/PLGA hybrid materials, with each material containing 0, 10, 20, 40, and 80 wt% of DBPs of PLGA. For biocompatibility test, NIH/3T3 mouse fibroblasts were cultured on the DBP/PLGA scaffold for 3 days. The inflammatory potential of PLGA was evaluated using messenger ribonucleic acid expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) on a human acute promyelocytic leukemic cell (HL-60). The in vivo response of DBP/PLGA film was compared with that of PLGA film implanted subcutaneously; the local inflammatory response was observed according to histology. The DBP/PLGA scaffold had no adverse effect on NIH/3T3 initial cell attachment and did not affect cell viability. DBP/PLGA films, especially PLGA films containing 80% DBP, elicited a significantly lower expression of IL-1beta and TNF-alpha from HL-60 cells than PLGA film alone. In vivo, DBP/PLGA film demonstrated a more favorable tissue response profile than PLGA film, with significantly less inflammation and fibrous capsule formation as below only 20% of DBP in PLGA film during implantation. This study shows that application of DBPs reduces the fibrous tissue encapsulation and foreign body giant cell response that commonly occurs at the interface of PLGA.

Influence of Matrix-Suspended Demineralized Bone on Osseous Repair Using a Critical-Sized Defect in the Rat (Rattus norvegicus) Calvarium

Demineralized freeze-dried bone (DFDB) in matrix form must be rehydrated with a carrier medium which allows for easy manipulation during periodontal surgery. The purpose of this study was to evaluate how human DFDB suspended in a polyol matrix affects new bone formation in the rat calvarium critical-sized defect (CSD) model. Fifty-five adult male Harlan Sprague-Dawley rats were assigned to 1 of 5 treatment groups: polyol, 100% DFDB, 47% DFDB/polyol, 47% DFDB, or an unfilled control. They were then placed into 8-m calvarial CSDs. The bone donor source company for the DFDB and DFDB/polyol groups was the same. Calvaria were harvested 10 weeks after surgery and evaluated histomorphometrically. The diameter of bone particles from the 3 groups containing DFDB was measured by scanning electron microscopy. There was no statistically significant difference in the percentage of bone fill between any of the groups, although the 100% DFDB group exhibited the most bone fill. The 47% DFDB/polyol and 47% DFDB groups had similar amounts of bone formation. The average size of the demineralized bone particles from the 100% DFDB group was significantly smaller than that of the other 2 groups containing DFDB. Adding a polyol to DFDB produced similar osseous regeneration in the rat calvarium defect model vs DFDB alone. Yet from a clinical standpoint, the polyol enhanced graft handling and stability. Graft particle size may have an effect on bone fill.

Effect of DBP/PLGA Hybrid Scaffold on Angiogenesis during the Repair of Calvarial Bone Defect

This study was designed to investigate the influence of demineralized bone particles (DBP)/PLGA hybrid scaffold on angiogenesis and osteogenesis in a calvarial defect model. DBP/PLGA scaffolds were manufactured by solvent casting/salt leaching method, and each scaffold contained 0, 10, 20, 40, and 80 wt% DBP of PLGA, respectively. A total of 34 rats were operated and bicortical holes were placed on their calvaria. The defects were filled with different ratio DBP/PLGA scaffolds. After 3, 7, 14, and 28 days, specimens were taken and, histologic, immunohistologic and RT-PCR analyses were carried out concerning number of vessels and density of regenerated bone, and angiogenic activation. On days 7, in all experimental groups, bone formation occurred in a direction from defected margin of calvarium to center of implanted scaffold and new vessel formation took place in front of the osteogenic regeneration front. We found that the 20 and 40 wt% DBP/PLGA scaffold was superior in its ability to regenerate new bone, induced more intensive formation of microvasculature and expressed in a higher level of osteocalcin mRNA than other groups.

Closure of Rabbit Calvarial Critical-Sized Defects Using Protective Composite Allogeneic and Alloplastic Bone Substitutes

This study evaluated the repair of critical-sized cranial vault defects in thirty New Zealand white rabbits using various allogeneic and alloplastic bone substitutes designed to provide mechanical protection to the brain as well as osteoinductivity. The strategies employed included demineralized bone matrix (DBM), a putty used in combination with a rigid resorbable plating system as a protective covering and calcium phosphate cement (CPC) combined with native partially purified bone morphogenetic protein (BMP). Bilateral critical-sized defects measuring 15 mm in diameter were created in the parietal bones of 30 adult male New Zealand white rabbits. They were divided into three groups with ten animals in each. Group 1 had one defect left unfilled as a control while autogenous bone was placed in the defect on the other side. In Group 2 a rigid resorbable copolymer membrane, Lactosorb (Lorenz Surgical, Jacksonville, Florida), was placed over both defects to cover them and protect the underlying tissues. The pericranial aspect of one defect was left unfilled while the other defect was filled with DBM putty. Group 3 had a CPC, Mimix (Lorenz Surgical, Jacksonville, Florida), placed into one of the defects while the defect on the other side was filled with the same CPC in combination with BMP in a concentration of 25 mg/mL. Bone healing was assessed clinically, radiographically, and histomorphometrically. All unfilled controlled defects, the defects covered with the resorbable Lactosorb membrane and those filled with calcium phosphate cement alone, healed with a fibrous scar. Defects reconstructed with DBM putty in combination with the resorbable Lactosorb membrane and calcium phosphate in combination with BMP healed with bone bridging the entire defect. This was obvious radiographically where the defects appeared completely filled with a dense radiopaque tissue. Histological analysis demonstrated that specimens where DBM putty was used in combination with the resorbable Lactosorb membrane had 67.7% new bone fill at 6 weeks and 84.0% at 12 weeks. Resorption of DBM particles was evidenced by the presence of osteoclastic activity and by the significant decrease in the size of the demineralized bone particles. In the calcium phosphate groups where BMP was added to the bioimplant there was 45.8% new bone formation at 12 weeks. The utilization of a composite consisting of DBM with resorbable Lactosorb membrane or a composite of calcium phosphate cement composite with BMP promoted complete closure of critical-sized calvarial defects in New Zealand white rabbits with viable new bone at 12 weeks. The complete bone bridging observed with these composites suggests that they could be used to enhance the protection of intracranial contents following craniofacial surgical procedures.

Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis–Menten kinetics with V=1278.7±38.4 nmol Pi/min/mg and K M=83.3±2.5 μM and V=473.9±18.9 nmol Pi/min/mg and K M=150.6±6.0 μM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium ( V=1084.7±32.5 nmol Pi/min/mg; and K M=377.8±11.3 μM) and magnesium ( V=1367.2±41.0 nmol Pi/min/mg and K M=595.3±17.8 μM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A 1, theophylline, thapsigargin, ethacrynic acid, P 1,P 5-(adenosine-5′)-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis. To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.

Biocompatibility and effect on osteogenesis of poly(ortho ester) compared to poly(DL‐lactic acid)

Implantation of demineralized bone induces new bone formation by the action of contained growth factors, of which bone morphogenetic proteins are of prime importance. A biodegradable polymer may be used as a carrier for demineralized bone particles or recombinant bone growth factors to prevent displacement of the implant, preserve its volume and shape, and assure sustained release of the incorporated active components. A polymer for this use should be biocompatible and completely absorbed without interfering with the osteogenesis. We investigated the host-tissue response and effect on demineralized bone-induced bone formation by two biodegradable polymers, a poly(ortho ester) and an amorphous low-molecular poly(DL-lactic acid). Both polymers had a plastic consistency, could easily be molded, and adhered well to the demineralized bone particles. Demineralized bone particles were implanted alone and in combination with each of the polymers in the abdominal muscles of 45 male Wistar rats. Four weeks after the operation the implants were recovered and subjected to 85Sr uptake analysis to quantify bone formation and histologic examination. The poly(ortho ester) provoked little inflammation; it was largely absorbed by 4 weeks, and no qualitative or quantitative effect on bone formation was found. The poly(DL-lactic acid) provoked a chronic inflammation with multinuclear giant cells, macrophages with engulfed material, and proliferating fibroblasts; part of the material was still present, and the bone formation was inhibited. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 257–263, 2000.

Use of demineralized bone as an osteoinductive orbital enucleation implant in the rabbit.

Implanted allogeneic demineralized bone particles (DBP) may be transformed into endochondral bone by inductive or conductive new bone formation. Using a rabbit model, enucleation or evisceration was performed, and DBP was implanted into the socket. Histology confirmed endochondral bone formation in both groups. A morphologic difference existed between enucleation and evisceration: compact bone was produced following evisceration, and cancellous bony spheres were formed following enucleation. Serial quantitative computed tomography (qCT) was performed to monitor mineral density and proved to be an ideal technique to monitor bone mineralization noninvasively in the postoperative orbit. Bone mineral density (BMD, mg/ml) increased approximately 300% in DBP implanted orbits when compared to controls at 6 weeks. The ideal mass/volume ratio of DBP to socket volume was 400 mg/ml. The rapidly formed bone is well vascularized and creates a stable integrated orbital implant, following enucleation and evisceration surgery. DBP implantation into the central orbit results in inductive bone formation with interesting potential in socket and orbit reconstruction.

Inhibition of induced endochondral bone development in caffeine‐treated rats

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGF beta were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.

Bone induction by demineralized bone particles: long-term observations of the implant-connective tissue interface.

Allogeneic demineralized bone (DB) powder was applied to subcutaneous pockets and cranial defects of rats and histological, histomorphometrical, and radiological evaluation was performed one to 52 weeks after implantation. In both type of implants cartilage formation was observed after 1 week between DB particles and in former vascular channels and cervices within the particles. Foci of bone formation were observed after 2 weeks in the center of the implant. Remineralization of DB particles only occurred in close contact with the new bone tissue by which they became incorporated, indicating that remineralization of DB is a continuation of a process which has started in live bone. At all times, the interface between implant and connective tissue of the host consisted of a layer of non-remineralized DB particles embedded in fibrous connective tissue. This rim was thinner at the dura-mater-side in the cranial implants, compared with the skin-side of the same implants as well as all sides of the subcutaneous implants. The rim became thinner with time but never disappeared completely. The outer contour of the mineralized implant sometimes ran right through a DB particle. A critical concentration of bone-inducing agent leaking from DB particles seems to be necessary for bone induction. We suggest that at the interface of the implant and loose connective tissue this concentration is never reached, which results in maintenance of the rim.