Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。方法 在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP-4成熟肽基因全长进行定点突变,将之重组入pET-3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。 结果 获得0.348 kb的BMP-4 DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。     

Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca²⁺ concentration ([Ca²⁺]_out) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca²⁺]_out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca²⁺]_out transiently increased the intracellular Ca²⁺ concentration ([Ca²⁺]_in) due to activation of the calcium-sensing receptor (CaSR). The Ca²⁺-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca²⁺]_out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca²⁺]_out enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca²⁺]_in and stimulation of Ca²⁺/calmodulin-dependent NFAT-signaling pathways.     

EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-7 IN Escherichia coli

Bone morphogenetic protein-7 (BMP-7) is a multifunctional cytokine of the transforming growth factor β superfamily, which induces bone formation and plays an important role during bone tissue repair and embryonic development. In this study, human BMP-7 (hBMP-7) cDNA was cloned and expressed in Escherichia coli, and its yield was approximately 30% of the total bacterial protein. After the bacteria were lysed by ultrasonication and repeated washing, inclusion bodies were extracted and dissolved using a high-strength denaturant. The monomer of rhBMP-7 was purified by ion-exchange chromatography, and the purity coefficient was approximately 96%. The protein was renatured with refolding buffers at different pH values. The renatured rhBMP-7 dimer protein in this study increased the alkaline phosphatase activity of NIH3T3 cells. This study may be helpful for the in vitro production and biomedical application of rhBMP-7 protein expressed in an E. coli expression system.     

The dynamic behaviours of protein BMP-2 on hydroxyapatite nanoparticles

The interaction between proteins and nanoscale inorganic particles is one of the most important topics in many fields. In this study, the dynamic behaviours of protein bone morphogenetic protein-2 (BMP-2) (with six different orientations) on hydroxyapatite (HAP) (001) surface were studied using the molecular dynamics and steered molecular dynamics simulation. The results show that the orientation of protein BMP-2 has obvious influence on its adsorption–desorption behaviours. Among the six systems studied in this article, system I exhibits the strongest interaction with the HAP (001) surface, and the number of the adsorbed residues is more than any one of the other five systems correspondingly. These findings suggest that there will be a preferential orientation when a protein is adsorbed onto a nanoscale interface. For protein BMP-2 interacting with the HAP (001) surface, the preferential orientation is the orientation in system I.     

Improved Anchorage of Ti6Al4V Orthopaedic Bone Implants through Oligonucleotide Mediated Immobilization of BMP-2 in Osteoporotic Rats

The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al₂O₃-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.     

Biotinated bone morphogenetic protein-2: In vivo and in vitro activity.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was biotinated, and the bioactivity of biotinated protein was assessed in vitro (alkaline phosphate induction in limb bud cells) and in vivo (osteoinduction in the rat ectopic assay). Amino-biotinated rhBMP-2 exhibited an increase in bioactivity whereas carboxy-biotinated rhBMP-2 did not exhibit any changes in bioactivity in vitro. Avidin inhibited the bioactivity of amino-biotinated but not carboxyl-biotinated rhBMP-2. Both amino- and carboxy-modified rhBMP-2 induced bone at an equivalent level to that of unmodified rhBMP-2 in vivo. The presence of avidin did not affect the osteoinductive activity of both types of biotinated rhBMP-2. The overall results indicated that binding to a large protein, avidin, might affect rhBMP-2 activity in vitro depending on the binding site; however, in vivo activity was unaffected by the avidin binding.     

BMP-2 Promotes Oral Squamous Carcinoma Cell Invasion by Inducing CCL5 Release

Bone morphogenetic protein-2 (BMP-2)-containing bone grafts are useful regenerative materials for oral and maxillofacial surgery; however, several in vitro and in vivo studies previously reported cancer progression-related adverse effects caused by BMP-2. In this study, by quantifying the rhBMP-2 content released from bone grafts, the rhBMP-2 concentration that did not show cytotoxicity in each cell line was determined and applied to the in vitro monoculture or coculture model in the invasion assay. Our results showed that 1 ng/ml rhBMP-2, while not affecting cancer cell viability, significantly increased the invasion ability of the cancer cells cocultured with fibroblasts. Cocultured medium with rhBMP-2 also contained increased levels of matrix metalloproteinases. rhBMP-2-treated cocultured fibroblasts did not show a prominent difference in mRNA expression profile. Some cytokines, however, were detected in the conditioned medium by a human cytokine antibody array. Among them, the cancer invasion-related factor CCL5 was quantified by ELISA. Interestingly, CCL5 neutralizing antibodies significantly reduced the invasion of oral cancer cells. In conclusion, our results suggest that 1 ng/ml rhBMP-2 may induce invasion of oral squamous cell carcinoma (OSCC) cells by CCL5 release in coculture models. Therefore, we propose that a careful clinical examination before the use of rhBMP-2-containing biomaterials is indispensable for using rhBMP-2 treatment to prevent cancer progression.     

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α_vβ₃ is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α_vβ₃ receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α_vβ₃ integrin/integrin-linked kinase/ERK signaling pathway.     

Bone morphogenetic protein activity preservation with extracorporeal irradiation- and liquid nitrogen freezing-treated recycled autografts for biological reconstruction in malignant bone tumor

Recycled autografts have been commonly used in biological reconstruction in conjunction with wide bone resection. Extracorporeal irradiation (ECIR) and freezing are the two major options for pretreating tumor-bearing autografts before transplant. This study, for the first time, compared the effects of these two techniques on bone morphogenetic protein (BMP)-2 activity. Bone tissue extracted from human femoral heads were treated through either ECIR at different doses (5000, 15,000, and 30,000 rad) or liquid nitrogen (LN) freezing for different durations (5, 10, and 15 min). The amount of BMP was analyzed through enzyme-linked immunosorbent assay (ELISA assay). Furthermore, we also used tandem mass spectrometry to analyze change of BMP-2 and BMP-7 expression after high dosage of irradiation (30,000 rad) and long-time of freezing (15 min). To directly evaluate the effect of ECIR or LN freezing treatment on the activity of BMP, commercial recombinant human BMP-2 (rhBMP-2) was added to the culture of human mesenchymal stem cells (hMSCs). The post-treatment activity of rhBMP-2 was quantitated by measuring the osteogenic differentiation of hMSCs with Alizarin Red S staining. Through Western blotting, the activation of the BMP signaling pathway by phospho-Smad antibodies was analyzed. Our results showed that post-treatment levels of BMP did not differ among the ECIR and LN freezing treatments in ELISA assay, but tandem mass spectrometry showed significantly lower expression of BMP-2 after 30,000 rad of irradiation. Both ECIR and freezing lowered the expression of regulatory factors involved in the BMP-activated signaling cascades and similar results were also observed in osteogenic differentiation of hMSCs. However, LN freezing preserved better bioactivity of rhBMP-2 whereas dosage-dependent declination was observed in ECIR groups. In conclusion, considering BMP-2 activity, LN freezing-treated autografts may result in a better osteoinduction outcomes than those treated using ECIR. Further investigation of the factors involved in bone formation is required.     

Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations

A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However, low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2 dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2 has potential clinical applications.     

材料表面化学刺激对纤连蛋白构象及细胞的影响

生物材料的表面化学性质通过改变吸附蛋白的构象,影响细胞粘附铺展,产生不同的细胞行为.采用金-硫自组装单分子层技术(alkanethiol self-assembled monolayers,SAMs)构建了不同化学基团(-NH2、-COOH、-OH和-CH3)修饰的基底材料表面.运用X射线光能质谱(XPS)和接触角仪表...     

Controlled release of injectable liposomal in situ gel loaded with recombinant human bone morphogenetic protein-2 for the repair of alveolar bone clefts in rabbits

Background and objective: The aim of the present study was to develop and examine a new non-invasive injectable graft for the repair of alveolar bone clefts using recombinant human bone morphogenetic protein-2 (rhBMP-2) encapsulated within injectable liposomal in situ gel (LIG).

Method: Different liposomal formulations loaded with rhBMP-2 were prepared, and the effects of the preparation methods and lipid content on the efficiency of rhBMP-2 encapsulation within the liposomes were studied. For the preparation of in situ gel, deacetylated gellan gum (DGG) was used, and the in vitro gelation characteristics of the gel were evaluated. In vivo pharmacokinetics and histology were also assessed. Critical size alveolar defects were surgically created in the maxillae of 30 New Zealand rabbits and treated with different injectable formulae, including rhBMP-2 liposomes and in situ gel (rhBMP-2-LIG).

Results: The results indicated that the prepared rhBMP-2-LIG prolonged the release and residence time of BMP-2 within rabbits for more than 7 days. Histomorphometric assessment showed 67% trabecular bone filling of the defects treated using this novel formula.

Conclusion: BMP-2-LIG is a promising delivery device for the repair of alveolar bone defects associated with cleft deformities.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.     

Expression and purification of recombinant human bone morphogenetic protein-7 (rhBMP-7) in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The polypeptide representing the mature part of human bone morphogenetic protein-7 (BMP-7) was cloned and efficiently expressed in Bacillus subtilis. Recombinant B. subtilis had a clear band for rhBMP-7, a homodimeric protein with an apparent molecular weight of 15.4 kDa and produced 350 pg rhBMP-7/mL of culture medium. The extracellular and intracellular rhBMP-7 was purified in two steps using a fast performance liquid chromatography (FPLC) system with an ion-exchange column and a gel filtration column. The extracellular rhBMP-7 had a purity of 57.1% and a yield of 58.8%, while the purity of the intracellular rhBMP-7 was 36.2% with a yield of 51.4%. The rhBMP-7 produced in this work also stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner, i.e. 2.5- and 8.9-fold at 100 and 300 ng rhBMP-7/mL, respectively, and showed intact biological activity.     

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold,Keratin, and BMP-2

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (K_D) of 1.8 × 10⁻⁴ M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (K_D = 1.1 × 10⁻⁷ M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (K_D = 3.2 × 10⁻⁵ M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.     

Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.