Calcium Phosphate-based Materials Research Articles

235. Evaluation of a nanosynthetic silicated calcium phosphate putty in a posterolateral rabbit spinal fusion model

BACKGROUND CONTEXT Providing surgeons with an “off the shelf” bone graft substitute to aid in bone healing is advantageous providing the materials participate in and facilitate the desired goal of fusion. Synthetic calcium phosphate-based (CaP) materials are excellent candidates considering their comparable composition to bone mineral however other factors have been shown to play a role in osteogenic behavior of these materials including surface chemistry and surface topography. The preclinical rabbit posterolateral fusion developed by Professor Boden provides a robust means to evaluate the in vivo performance of graft materials in this challenging indication. PURPOSE This study aims to evaluate the in-vivo response of a nanosynthetic silicated CaP combined with autograft compared to autograft alone or a collagen-biphasic CaP combined with autograft in a rabbit spinal fusion model. STUDY DESIGN/SETTING Preclinical animal model. PATIENT SAMPLE N/A OUTCOME MEASURES N/A METHODS Following ethical approval, 70 skeletally mature female NZ white rabbits were divided into three groups: Group 1: Autograft alone (positive control); Group 2: Nanosynthetic Silicated CaP Putty + Autograft (50/50); Group 3: Collagen-Biphasic CaP Putty + Autograft (50/50). A single level posterolateral intertransverse process lumbar fusion was performed bilaterally after decortication of the transverse processes with a high-speed burr. Iliac crest autograft was harvested bilaterally in the positive control group and 2cc placed in the posterolateral gutters. The synthetic materials (1cc) were combined with autograft (1cc) and implanted. Fusion assessment at day 0 (n=2), 6, 9 weeks (n=5), 12 weeks (n=8) and 26 weeks (n=5) included harvest, manual palpation, radiographic assessment, micro-CT, non-destructive range of motion testing (12 weeks only) and decalcified paraffin and pmMA histology; no positive control group (autograft alone) was included at the 26-week time point. RESULTS The positive control group, autograft alone, performed consistent with this model in all endpoints. In the synthetic groups combined with autograft (Groups 2 and 3), radiographic incorporation was observed at the host transverse processes at 6 weeks and further into the fusion with time. Graft resorption and new bone formation was noted in the radiographs as well as the micro-computed tomography sections. Manual palpation was 7/8 at 12 weeks in the Nanosynthetic Silicated CaP Putty + Autograft group compared to 4/8 for the Collagen-Biphasic CaP Putty + Autograft. Histology showed normal healing and increased bone remodeling/formation with Nanosynthetic Silicated CaP Putty + Autograft at all timepoints compared to the Collagen-Biphasic CaP Putty + Autograft. New bone formed directly on the Nanosynthetic Silicated CaP material surface within the fusion mass while this was not a feature of the Collagen-Biphasic CaP material. By 26 weeks histology revealed consistent bridging of bone between the transverse processes in the Nanosynthetic Silicated CaP Putty + Autograft group, but this was not observed in the Collagen-Biphasic CaP group. CONCLUSIONS Developing nanosynthetic silicated CaP bone grafts that aim to mimic the size, structure and chemistry of bone mineral can eliminate the need for excessive autograft harvest and results from this preclinical study supports their effective use in spinal fusion. FDA DEVICE/DRUG STATUS Osteo 3 (the Nanosynthetic Silicated CaP Putty) (Investigational/Not approved)

Injectable Functional Biomaterials for Minimally Invasive Surgery.

Injectable materials represent very attractive ready-to-use biomaterials for application in minimally invasive surgical procedures. It is shown that this approach to treat, for example, vertebral fracture, craniofacial defects, or tumor resection has significant clinical potential in the biomedical field. In the last four decades, calcium phosphate cements have been widely used as injectable materials for orthopedic surgery due to their excellent properties in terms of biocompatibility and osteoconductivity. However, few clinical studies have demonstrated certain weaknesses of these cements, which include high viscosity, long degradation time, and difficulties being manipulated. To overcome these limitations, the use of sol-gel technology has been investigated, which has shown good results for synthesis of injectable calcium phosphate-based materials. In the last few decades, injectable hydrogels have gained increasing attention owing to their structural similarities with the extracellular matrix, easy process conditions, and potential applications in minimally invasive surgery. However, the need to protect cells during injection leads to the development of double network injectable hydrogels that are capable of being cross-linked in situ. This review will provide the current state of the art and recent advances in the field of injectable biomaterials for minimally invasive surgery.

3D Printing of Bone Grafts for Cleft Alveolar Osteoplasty - In vivo Evaluation in a Preclinical Model.

One of the most common hereditary craniofacial anomalies in humans are cleft lip and cleft alveolar bone with or without cleft palate. Current clinical practice, the augmentation of the persisting alveolar bone defect by using autologous bone grafts, has considerable disadvantages motivating to an intensive search for alternatives. We developed a novel therapy concept based on 3D printing of biodegradable calcium phosphate-based materials and integration of osteogenic cells allowing fabrication of patient-specific, tissue-engineered bone grafts. Objective of the present study was the in vivo evaluation of implants in a rat alveolar cleft model. Scaffolds were designed according to the defect’s geometry with two different pore designs (60° and 30° rotated layer orientation) and produced by extrusion-based 3D plotting of a pasty calcium phosphate cement. The scaffolds filled into the artificial bone defect in the palate of adult Lewis rats, showing a good support. Half of the scaffolds were colonized with rat mesenchymal stromal cells (rMSC) prior to implantation. After 6 and 12 weeks, remaining defect width and bone formation were quantified histologically and by microCT. The results revealed excellent osteoconductive properties of the scaffolds, a significant influence of the pore geometry (60° > 30°), but no enhanced defect healing by pre-colonization with rMSC.

Magnesium Silicate Bioceramics for Bone Regeneration: A Review

With the increase in need of ideal bone graft materials, magnesium silicate has been explored as resorbable bioceramics. Calcium phosphate-based bioceramics are well studied and being implemented for several orthopaedic applications as they mimic the chemistry of the natural bone. Although extensively used, these materials do not satisfy all the essential requirements of an ideal temporary bone replacement material. Materials, such as tricalcium phosphate (TCP) and hydroxyapatite (HA), have low solubility and often the resorption rate is quite slow when implanted in vivo. The research on magnesium silicate for bone regenerative application is quite relatively young and a new area compared to traditional calcium phosphate-based materials. Although limited research findings have been reported, it is believed that magnesium silicate-based bioceramics may be an alternative to calcium phosphate for bone tissue engineering applications. Thus in this review, we have highlighted the importance of magnesium silicate bioceramics and compared with existing calcium phosphate ceramics. We have also analysed the future directions and the need for clinical implementations.

Setting Properties of Calcium Phosphate Bone Cement

Calcium phosphate cements (CPCs) are widely used in bone repair. CPCs show many advantages comparing to other calcium phosphate-based materials because of its chemical similarity to the mineral constituent of natural bone. This work describes a new apatitic calcium phosphate cement composition with a Ca/P ratio = 1.38, by mixing two components which were prepared by wet chemical synthesis methods. One component is chemically synthesized by mixing α-tricalcium phosphate (αTCP, Ca3(PO4)2) and HA (Ca10(PO4)6OH)2), while the second component is again a chemically synthesized single phase DCPD (CaHPO4.2H2O) powder. The sitting solution used is 3 wt% Na2HPO4. 2H2O dissolved in distilled water with a liquid to powder ratio L/P = 0.4. The cement was characterized in terms of setting time, compressive strength. XRD and SEM techniques were employed to evaluate the phase composition and surface morphology of the cement, the powder XRD data shows that the cement sample soaked in water has transformed into calcium deficient hydroxyapatite. The cement showed a fast setting time (14 min), this cement also showed a high compressive strength (35 MPa).

Bioactive glass/hydroxyapatite- containing electrospun poly (ε-Caprolactone) composite nanofibers for bone tissue engineering

In this study, bioactive glass and hydroxyapatite (HA)-containing poly(e-caprolactone) (PCL) nanocomposite fiber mats were fabricated through electrospinning. For this purpose, microscale bioactive glass (silicate-based 45S5 and borate-based 13-93B3 compositions) or HA particles (at 10 wt%) were incorporated into the PCL matrix. The fabricated biocomposite fibers were investigated in terms of morphological and chemical properties. An in vitro mineralization assay in simulated body fluid was performed to understand the capability of the composite electrospun fibers to induce the formation of hydroxycarbonate apatite. Results showed that the diameter of the electrospun PCL-based fibrous scaffolds increased by the inclusion of bioactive glass or HA particles. All of the fibrous mats prepared in the study showed hydrophobic character. Relatively high contact angles (> 90°) obtained for fibrous scaffolds was attributed to the high porosity and surface roughness. Bioactive glass or HA addition to the PCL matrix enhanced the bioactivity of the fibrous scaffolds. The deposition rate of calcium phosphate-based material precipitates was higher on the surface of HA-containing samples compared to bioactive glass-containing PCL scaffolds. Additionally, mineralization ability of borate-based 13-93B3 glass-containing samples was higher compared to 45S5 glass-containing PCL fibers. The biocomposite fibrous scaffolds prepared in the study may find applications in wound healing as wound dressing and in bone tissue engineering.

Osteoblast Cell Response to Naturally Derived Calcium Phosphate-Based Materials.

The demand of calcium phosphate bioceramics for biomedical applications is constantly increasing. Efficient and cost-effective production can be achieved using naturally derived materials. In this work, calcium phosphate powders, obtained from dolomitic marble and Mytilus galloprovincialis seashells by a previously reported and improved Rathje method were used to fabricate microporous pellets through cold isostatic pressing followed by sintering at 1200 °C. The interaction of the developed materials with MC3T3-E1 pre-osteoblasts was explored in terms of cell adhesion, morphology, viability, proliferation, and differentiation to evaluate their potential for bone regeneration. Results showed appropriate cell adhesion and high viability without distinguishable differences in the morphological features. Likewise, the pre-osteoblast proliferation overtime on both naturally derived calcium phosphate materials showed a statistically significant increase comparable to that of commercial hydroxyapatite, used as reference material. Furthermore, evaluation of the intracellular alkaline phosphatase activity and collagen synthesis and deposition, used as markers of the osteogenic ability of these bioceramics, revealed that all samples promoted pre-osteoblast differentiation. However, a seashell-derived ceramic demonstrated a higher efficacy in inducing cell differentiation, almost equivalent to that of the commercial hydroxyapatite. Therefore, data obtained demonstrate that this naturally sourced calcium-phosphate material holds promise for applications in bone tissue regeneration.

Silicon-doped hydroxyapatite prepared by a thermal technique for hard tissue engineering applications

Hydroxyapatite (HA, Ca5(PO4)3OH) has been extensively used for bone implantation due to its similarity to the mineral component of bone, which makes it strongly osteoconductive. However, HA has low resorbability, and it is difficult to replace by a newly regenerated bone. Si doping can enhance the resorbability of HA by modifying its crystal structure. Here, we developed a simple thermal technique for preparing Si-doped HA from silica (SiO2) and HA precursors, both of which are inexpensive and commercially available. This method included the physical binding of SiO2 and HA particles, followed by pressing and sintering the mixture at an elevated temperature, which enhanced the atomic diffusion of Si into HA unit cells. We also evaluated the simulated body fluid (SBF) activity of the Si-doped HA prepared by this technique and showed that it significantly had higher resorbability and mineralizing potential compared to the pure HA. Our experimental design including, the individual precipitation and resorption assays enabled us to explain the mechanism behind the improved activity of Si-doped HA in SBF. This was attributed to the formation of new phases, such as β-tricalcium phosphate (β-TCP) and calcium silicate (Ca2SiO4) with higher solubility than HA on the SiO2-contating HA during the sintering stage. This can provide some guidelines for designing new calcium phosphate-based materials for hard tissue engineering applications.

Complications after craniofacial reconstruction with calcium phosphate cements: a case report and review of the literature.

Among different graft materials for craniofacial reconstruction, calcium phosphate cements have the advantages of alloplastic grafts and wide use. The authors report a case of foreign body reaction following frontal reconstruction with JectOS (an injectable calcium orthophosphate cement; Kasios) and reviewed the literature on complications of this material after craniofacial reconstruction from 2002 to 2017. Complications were categorized into two groups: immunologic reactions (consisting of seroma collection, chronic sinus mucosa swelling, and foreign body reaction) and non-immune events (infection, fragmentation, and ejection). It is wise to use calcium phosphate-based material only in selected cases with small defects, and long-term follow-up is needed to observe their consequences.

Osseoconductive and Corrosion-Inhibiting Plasma-Sprayed Calcium Phosphate Coatings for Metallic Medical Implants

During the last several decades, research into bioceramic coatings for medical implants has emerged as a hot topic among materials scientists and clinical practitioners alike. In particular, today, calcium phosphate-based bioceramic materials are ubiquitously used in clinical applications to coat the stems of metallic endoprosthetic hips as well as the surfaces of dental root implants. Such implants frequently consist of titanium alloys, CoCrMo alloy, or austenitic surgical stainless steels, and aim at replacing lost body parts or restoring functions to diseased or damaged tissues of the human body. In addition, besides such inherently corrosion-resistant metals, increasingly, biodegradable metals such as magnesium alloys are being researched for osseosynthetic devices and coronary stents both of which are intended to remain in the human body for only a short time. Biocompatible coatings provide not only vital biological functions by supporting osseoconductivity but may serve also to protect the metallic parts of implants from corrosion in the aggressive metabolic environment. Moreover, the essential properties of hydroxylapatite-based bioceramic coatings including their in vitro alteration in contact with simulated body fluids will be addressed in this current review paper. In addition, a paradigmatic shift is suggested towards the development of transition metal-substituted calcium hexa-orthophosphates with the NaSiCON (Na superionic conductor) structure to be used for implant coatings with superior degradation resistance in the corrosive body environment and with pronounced ionic conductivity that might be utilized in novel devices for electrical bone growth stimulation.

A Short Overview on the Biomedical Applications of Silica, Alumina and Calcium Phosphate-based Nanostructured Materials.

This article reviews the use of silica, alumina and calcium phosphate-based nanostructured materials with biomedical applications. A short introduction on the use of the materials in Science, Nanotechnology and Health is included followed by a revision of each of the selected materials. A description of the principal synthetic methods used in the preparation of the materials in nanostructured form is included. The most widely used applications in biomedicine are reviewed including, for example drug-delivery, bone regeneration, imaging, sensoring amongst others. Finally, a short description of the toxicity and cytotoxicity associated with each of the materials of this revision is presented. This short literature revision serves to demonstrate the very promising future ahead of nanosystems based on silica, alumina and calcium phosphate for biological and biomedical applications.

Micropore-induced capillarity enhances bone distribution in vivo in biphasic calcium phosphate scaffolds.

The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVF‾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVF‾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects. The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100μm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (<20μm) also enhances growth, but there is no consensus on the controlling mechanisms. In previous in vitro work, we suggested that micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly enhances the bone distribution in the scaffold. The results will impact the design of scaffolds to better exploit capillarity and improve treatments for large and load-bearing bone defects.

Magnesium Phosphate Cement Systems for Hard Tissue Applications: A Review.

In the search for more ideal bone graft materials for clinical application, the investigation into ceramic bone cements or bone void filler is ongoing. Calcium phosphate-based materials have been widely explored and implemented for medical use in bone defect repair. Such materials are an excellent choice because the implant mimics the natural chemistry of mineralized bone matrix and in injectable cement form, can be implemented with relative ease. However, of the available calcium phosphate cements, none fully meet the ideal standard, displaying low strengths and acidic setting reactions or slow setting times, and are often very slow to resorb in vivo. The study of magnesium phosphates for bone cements is a relatively new field compared to traditional calcium phosphate bone cements. Although reports are more limited, preliminary studies have shown that magnesium phosphate cements (MPC) may be a strong alternative to calcium phosphates for certain applications. The goal of the present publication is to review the history and achievements of magnesium phosphate-based cements or bone void fillers to date, assess how these cements compare with calcium phosphate competitors and to analyze the future directions and outlook for the research, development, and clinical implementation of these cements.

Approaches to the fabrication of calcium phosphate-based porous materials for bone tissue regeneration

This paper reviews advances in the fabrication of calcium phosphate materials for injured bone tissue regeneration. We examine the key features of rapid prototyping for the fabrication of porous ceramic scaffolds with tailored architectures, the technology of biopolymer-based composite materials reinforced with calcium phosphate particles, and the fabrication of porous scaffolds via cement route.

A new iron calcium phosphate material to improve the osteoconductive properties of a biodegradable ceramic: a study in rabbit calvaria

β-tricalcium phosphate (β-TCP) is an osteoconductive and biodegradable material used in bone regeneration procedures, while iron has been suggested as a tool to improve the biological performance of calcium phosphate-based materials. However, the mechanisms of interaction between these materials and human cells are not fully understood. In order to clarify this relationship, we have studied the iron role in β-TCP ceramics. Iron-containing β-TCPs were prepared by replacing CaCO3 with C6H5FeO7 at different molar ratios. X-ray diffraction analysis indicated the occurrence of β-TCP as the sole phase in the pure β-TCP and iron-containing ceramics. The incorporation of iron ions in the β-TCP lattice decreased the specific surface area as the pore size was shifted toward meso- and/or macropores. Furthermore, the human osteoblastlike cell line MG-63 was cultured onto the ceramics to determine cell proliferation and viability, and it was observed that the iron-β-TCP ceramics have better cytocompatibility than pure β-TCP.Finally, in vivo assays were performed using rabbit calvaria as a bone model. The scaffolds were implanted for 8 and 12 weeks in the defects created in the skullcap with pure β-TCP as the control. The in vivo behavior, in terms of new bone formed, degradation, and residual graft material were investigated using sequential histological evaluations and histomorphometric analysis. The in vivo implantation of the ceramics showed enhanced bone tissue formation and scaffold degradation for iron-β-TCPs. Thus, iron appears to be a useful tool to enhance the osteoconductive properties of calcium phosphate ceramics.

One-step synthesized calcium phosphate-based material for the removal of alizarin S dye from aqueous solutions: isothermal, kinetics, and thermodynamics studies

Calcium phosphate hydroxyapatite (Ca-Hap) synthesized from CaCO3 and H3PO5, it was characterized by scanning electron microscopy, Fourier transform infrared, and X-ray diffraction. The Ca-Hap was used for the removal of Alizarin Red S dye from its aqueous solution. The kinetics, equilibrium, and thermodynamic of the adsorption of the dye onto the Ca-Hap were investigated. The effects of contact time, initial dye concentration, pH as well as temperature on adsorption capacity of Ca-Hap were studied. Experimental data were analyzed using six model equations: Langmuir, Freudlinch, Redlich–Peterson, Temkin, Dubinin–Radushkevich, and Sips isotherms and it was found that the data fitted well with Sips and Dubinin–Radushkevich isotherm models. Pseudo-first-order, pseudo-second-order, Elovic, and Avrami kinetic models were used to test the experimental data in order to elucidate the kinetic adsorption process and it was found that pseudo-second-order model best fit the data. The calculated thermodynamics parameters (∆G°, ∆H° and ∆S°) indicated that the process is spontaneous and endothermic in nature.

Calcium phosphate nanocoatings and nanocomposites, part I: recent developments and advancements in tissue engineering and bioimaging.

A number of materials have been applied as implant coatings and as tissue regeneration materials. Calcium phosphate holds a special consideration, due to its chemical similarity to human bone and, most importantly, its dissolution characteristics, which allow for bone growth and regeneration. The applications of molecular and nanoscale-based biological materials have been and will continue to play an ever increasing role in enhancing and improving the osseointegration of dental and orthopedic implants. More recently, extensive research efforts have been focused on the development and applications of fluorescent nanoparticles and nanocoatings for in vivo imaging and diagnostics as well as devising methods of adding luminescent or fluorescent capabilities to enhance the in vivo functionality of calcium phosphate-based biomedical materials.

Effect of the deposition temperature on corrosion resistance and biocompatibility of the hydroxyapatite coatings

Hydroxyapatite (HAP) ceramics belong to a class of calcium phosphate-based materials, which have been widely used as coatings on titanium medical implants in order to improve bone fixation and thus to increase the lifetime of the implant. In this study, HAP coatings were deposited from pure HAP targets on Ti6Al4V substrates using the radio-frequency magnetron sputtering technique at substrate temperatures ranging from 400 to 800°C. The surface morphology and the crystallographic structure of the films were investigated by atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). The corrosion resistance of the coatings in saliva solution at 37°C was evaluated by potentiodynamic polarization. Additionally, the human osteosarcoma cell line (MG-63) was used to test the biocompatibility of the coatings. The results showed that all of the coatings grown uniformly and that the increasing substrate temperature induced an increase in their crystallinity. Corrosion performance of the coatings was improved with the increase of the substrate temperature from 400°C to 800°C. Furthermore, all the coatings support the attachment and growth of the osteosarcoma cells with regard to the in vitro test findings.

Migration of Alloplastic Bone Graft Material in Infected Conditions: A Case Study and Animal Experiment

Distant migration associated with sinus lifting procedures has not been investigated. In the present study, a case of distant migration of graft material was observed, and the potential mechanisms of migration were analyzed using material analysis and in vivo experiments. The migrated graft material was biphasic calcium phosphate-based alloplastic material (BCP), and its physical properties were compared with those of xenogenic material (Bio-Oss). The comparisons of the physical properties were performed using scanning electronic microscopic, x-ray diffraction, and Fourier-transform infrared absorbance spectra analysis. The comparative graft migration study was performed using the subcutaneous pocket model in rats (n = 10). The clinical case was analyzed by histologic section and energy dispersive x-ray (EDX) microanalysis. The observed diffraction patterns from the Bio-Oss revealed characteristic diffractions for the hydroxyapatite phase, and those from the BCP revealed additional diffractions that could be assigned to the tricalcium phosphate phase. In the animal model, the graft migration distances observed in the BCP group were significantly greater than those observed in the Bio-Oss group (P = .012). In the clinical case, the lymphatic vessels of the submandibular gland contained foreign materials that were morphologically similar to those of the maxillary sinus. EDX microanalysis revealed that the particles in the lymphatic vessels exhibited calcium concentrations that were approximately 200 times greater than those in the adjacent glandular tissue. In the present study, BCP-based sinus grafts had migrated into the submandibular glandular area by way of the lymphatic chain in the presented clinical case.

Evaluation of a β‐Calcium Metaphosphate Bone Graft Containing Bone Morphogenetic Protein‐7 in Rabbit Maxillary Defects

Calcium phosphate-based materials have been widely used as bone substitutes and more recently are being exploited together with growth factors as bone tissue engineering scaffolds regulating cell behavior. The aim of this study is to evaluate the in vitro and in vivo response to a newly developed calcium metaphosphate (CMP) bone graft, with and without bone-stimulating growth factor. Porous scaffolds of CMP were developed and extensively tested in vitro. Subsequently, CMP grafts with osteogenic protein-1 (OP-1) (test) and without OP-1 (control) were implanted into experimental rabbit maxillary bone defects. Animals were sacrificed at 2, 4, and 8 weeks, and samples were examined with microcomputed tomography (micro-CT) and processed for histomorphometric analysis. At 8 weeks, the scaffolds containing OP-1 induced greater bone formation (P = 0.018) than CMP alone, based on histomorphometric evaluation (percentage bone area: test: 57.1 ± 5.6; control: 49.4 ± 7.7) and micro-CT analysis (percentage bone volume density: test: 63.46 ± 5.61; control: 51.20 ± 6.71). Thus, these data indicated that both test and control CMP grafts showed a good degree of bone formation. Furthermore, the CMP materials showed signs of resorption from 4 weeks, and no graft materials were observed at 8 weeks. In vitro, the OP-1 loaded graft demonstrated a release profile and bioactivity over a 28-day period. In vivo testing confirmed enhanced bone formation of the OP-1 loaded graft after 8 weeks of healing.