"Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions.

Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

The construction of a microenvironment with the vascular network by co-culturing fibroblasts and endothelial cells.

Introduction: Extracellular matrix (ECM) synthesis and deposition in fibroblasts, and vascularization via endothelial cells are essential for successful tissue regeneration. Fibroblasts can produce both ECM, physical support for maintaining homeostasis, and bioactive molecules, such as growth factors and cytokines. Endothelial cells can secrete growth factors and form vascular networks that enable the supply of nutrients and oxygen and remove metabolic products. Methods: In this study, we focused on combining Human Periodontal Ligament Fibroblasts (HPLF) and Human Umbilical Vein Endothelial Cells (HUVEC) for tissue regeneration in clinical applications. Results: The fibroblastic and angiogenic phenotypes were promoted in co-culture with HPLF and HUVEC at a ratio of 1:1 compared to HPLF or HUVEC mono-culture. The gene expression of ECM components and angiogenesis-related factors was also enhanced by HPLF/HUVEC co-culture. Despite an apparent increase in the expression of angiogenic factors, the levels of secreted growth factors decreased under co-culture conditions. These data suggest that ECM constructed by HPLF and HUVEC would act as a storage site for growth factors, which can later be released. Our results showed that cell-to-cell interactions between HPLF and HUVEC enhanced collagen synthesis and endothelial network formation, leading to the creation of highly vascularized constructs for periodontal tissue regeneration. Conclusion: Successful periodontal tissue regeneration requires microenvironmental reconstruction and vascularization, which can be achieved using a co-culture system. In the present study, we found that fibroblastic and angiogenic phenotypes were enhanced by the co-culture of HPLF and HUVEC. The optimal culture conditions (1:1) could potentially accelerate tissue engineering, including ECM synthesis and EC tube formation, and these approaches can improve therapeutic efficacy after transplantation.

A Novel Control Method of Enterococcus faecalis by Co-Treatment with Protamine and Calcium Hydroxide.

Enterococcus faecalis (E. faecalis), a gram-positive facultative anaerobic bacterium, is likely to survive root canal treatment because of its extremely high alkaline tolerance, which may contribute to the refractory nature of apical periodontitis (AP). In this study, protamine was combined with calcium hydroxide to evaluate its efficacy in killing E. faecalis. First, the antibacterial activity of protamine against E. faecalis was investigated. Protamine reduced the E. faecalis growth rate at concentrations above the MIC (250 µg/mL), but was not bactericidal at any of the concentrations tested. Next, we investigated the calcium hydroxide tolerance of E. faecalis, using a 10% 310 medium, adjusted for pH by adding a calcium hydroxide solution. The results showed that E. faecalis could survive and proliferate in alkaline environments up to pH 10. However, the complete killing of E. faecalis was observed when protamine (250 µg/mL) was added. In addition, compared with treatment with protamine and calcium hydroxide alone, membrane damage and internalization of protamine into the cytoplasm of E. faecalis were enhanced. Therefore, the synergistic increase in antibacterial activity may be related to the action of both antimicrobial agents on the cell membrane. In conclusion, co-treatment with protamine and calcium hydroxide seems to be very effective in sterilizing E. faecalis, and has the potential to provide a novel control method against E. faecalis for root canal treatment.

Wnt/ß-Catenin Signaling Inhibits Osteogenic Differentiation in Human Periodontal Ligament Fibroblasts.

The periodontal ligament is a collagenous tissue that is important for maintaining the homeostasis of cementum and alveolar bone. In tendon cells, Wnt/ß-catenin signaling has been reported to regulate the expression level of Scleraxis (Scx) and Mohawk Homeobox (Mkx) gene and maintain the tissue homeostasis, while its role in the periodontal ligament is unclear. The aim of this study was to investigate the effects of Wnt/ß-catenin signaling induced by Wnt-3a stimulation on the inhibition of osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs). During osteogenic differentiation of HPLFs, they formed bone nodules independently of alkaline phosphatase (ALP) activity. After stimulation of Wnt-3a, the expression of ß-catenin increased, and nuclear translocation of ß-catenin was observed. These data indicate that Wnt-3a activated Wnt/ß-catenin signaling. Furthermore, the stimulation of Wnt-3a inhibited the bone nodule formation and suppressed the expression of osteogenic differentiation-related genes such as Runx2, Osteopontin and Osteocalcin, and upregulated the gene expression of Type-I collagen and Periostin (Postn). Scx may be involved in the suppression of osteogenic differentiation in HPLFs. In conclusion, Wnt/ß-catenin signaling may be an important signaling pathway that inhibits the osteogenic differentiation in HPLFs by the upregulation of Scx gene expression and downregulation of osteogenic differentiation-related genes.

Preparation of antimicrobial calcium phosphate/protamine composite powders with fluoride ions using octacalcium phosphate.

Calcium phosphates are key biomaterials in dental treatment and bone regeneration. Biomaterials must exhibit antibacterial properties to prevent microbial infection in implantation frameworks. Previously, we developed various types of calcium phosphate powders (amorphous calcium phosphate, octacalcium phosphate (OCP), dicalcium phosphate anhydrate, and hydroxyapatite) with adsorbed protamine (which is a protein with antibacterial property) and confirmed their antibacterial property. In this study, as foundational research for the development of novel oral care materials, we synthesized calcium phosphate composite powders from three starting materials: i) OCP, which intercalates organic compounds, ii) protamine, which has antibacterial properties, and iii) F- ion, which promotes the formation of apatite crystals. Through investigating the preparation concentration of the F- ions and their loading into OCP, it was found that more F- ion could be loaded at higher concentrations regardless of the loading method. It was also observed that the higher the preparation concentration, the more the OCP converted to fluorapatite. The synthesized calcium phosphate composite powders were evaluated for biocompatibility through proliferation of MG-63 cells, with none of the powders exhibiting any growth inhibition. Antimicrobial tests showed that the calcium phosphate composite powders synthesized with protamine and F- ion by precipitation had enhanced antimicrobial properties than those synthesized by protamine adsorption. Thus, the calcium phosphate composite powder prepared from OCP, protamine, and F- ion forms the basis for promising antimicrobial biomaterials. Graphical abstract.

Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor.

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Regulating size of silver nanoparticles on calcium carbonate via ultrasonic spray for effective antibacterial efficacy and sustained release.

Calcium carbonate is used as bone-filling material due to its good biocompatibility, bioactivity, and bioabsorbability, but the prevalence of infectious complications associated with calcium carbonate has created a persisting challenge in the treatment of bone defect. Therefore, this greatly necessitate the need to endow calcium carbonate with antibacterial properties. In this study, calcium carbonate powders loaded with silver nanoparticles (Ag-CaCO3) were prepared in attempt to serve as a novel antibacterial inorganic filler material. This objective was achieved using ultrasonic spray-pyrolysis (USSP) route to produce Ag-CaCO3 with 1, 5 and 10 mol% silver. The size of silver nanoparticles on CaCO3 microspheres could be regulated by adjusting silver concentration to facilitate effective release of Ag+ ions. This was demonstrated in Ag-CaCO3 (1), where the lowest silver content at 1 mol% achieved the highest Ag+ ions release over 28 days. This in turn gave rise to effective antibacterial efficiency against Staphylococcus aureus and Escherichia coli. Furthermore, CaCO3 (1) could also support osteoblast-like cells (MG-63) at a cell viability of 80%. Overall, this work extends the capabilities in employing USSP to produce inorganic filler materials with sustained antibacterial properties, bringing one step closer to the development of antibacterial products.

Zoledronic Acid-Loaded ß-TCP Inhibits Tumor Proliferation and Osteoclast Activation: Development of a Functional Bone Substitute for an Efficient Osteosarcoma Treatment.

Osteosarcoma has a poor survival rate due to relapse and metastasis. Zoledronic acid (ZOL), an anti-resorptive and anti-tumor agent, is used for treating osteosarcoma. Delivery of ZOL to the target region is difficult due to its high binding affinity to bone minerals. This study developed a novel treatment for osteosarcoma by delivering ZOL to the target region locally and sustainably. In this study, we fabricated a novel bone substitute by loading ZOL on ß-tricalcium phosphate (ß-TCP). The ZOL-loaded ß-TCP (ZOL/ß-TCP) would be expected to express the inhibitory effects via both bound-ZOL (bound to ß-TCP) and free-ZOL (release from ZOL/ß-TCP). To explore the ability to release ZOL from the ZOL/ß-TCP, the amount of released ZOL was measured. The released profile indicates that a small amount of ZOL was released, and most of it remained on the ß-TCP. Our data showed that ZOL/ß-TCP could successfully express the effects of ZOL via both bound-ZOL and free-ZOL. In addition, we examined the biological effects of bound/free-ZOL using osteosarcoma and osteoclasts (target cells). The results showed that two states of ZOL (bound/free) inhibit target cell activities. As a result, ZOL/ß-TCP is a promising candidate for application as a novel bone substitute.

Potential Application of Protamine for Antimicrobial Biomaterials in Bone Tissue Engineering.

Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal activity. Antimicrobial peptides and proteins (AMPs), alternative antibacterial agents, are expected to overcome the bacterial resistance. The aim of this study was to develop a new intelligent material in bone tissue engineering based on protamine-loaded hydroxyapatite (protamine/HAp) that uses AMPs rather than antibiotics. It was found that the adsorption of protamine to HAp followed the Langmuir adsorption model and was due to electrostatic and/or hydrophobic interactions. In vitro bacterial adhesion and growth on protamine/HAp was inhibited in a protamine dose-dependent manner. Adherent bacteria exhibited an aberrant morphology for high dosages of protamine/HAp, resulting in the formation of large aggregates and disintegration of the membrane. The released protamine from protamine/HAp also prevented the growth of planktonic bacteria in vitro. However, a high dosage of protamine from powders at loading concentrations over 1000 µg·mL-1 induced a cytotoxic effect in vitro, although those exhibited no apparent cytotoxicity in vivo. These data revealed that protamine/HAp (less than 1000 µg·mL-1) had both antimicrobial activity and biocompatibility and can be applied for bone substitutes in orthopedic fields.

Bactericidal and Bioresorbable Calcium Phosphate Cements Fabricated by Silver-Containing Tricalcium Phosphate Microspheres.

Bacterial adhesion to the calcium phosphate surface is a serious problem in surgery. To prevent bacterial infection, the development of calcium-phosphate cements (CPCs) with bactericidal properties is indispensable. The aim of this study was to fabricate antibacterial CPCs and evaluate their biological properties. Silver-containing tricalcium phosphate (Ag-TCP) microspheres consisting of α/ß-TCP phases were synthesized by an ultrasonic spray-pyrolysis technique. The powders prepared were mixed with the setting liquid to fabricate the CPCs. The resulting cements consisting of ß-TCP and hydroxyapatite had a porous structure and wash-out resistance. Additionally, silver and calcium ions could be released into the culture medium from Ag-TCP cements for a long time accompanied by the dissolution of TCP. These data showed the bioresorbability of the Ag-TCP cement. In vitro antibacterial evaluation demonstrated that both released and immobilized silver suppressed the growth of bacteria and prevented bacterial adhesion to the surface of CPCs. Furthermore, histological evaluation by implantation of Ag-TCP cements into rabbit tibiae exhibited abundant bone apposition on the cement without inflammatory responses. These results showed that Ag-TCP cement has a good antibacterial property and good biocompatibility. The present Ag-TCP cements are promising for bone tissue engineering and may be used as antibacterial biomaterials.

Correction: Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy.

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Pigs with δ-sarcoglycan deficiency exhibit traits of genetic cardiomyopathy.

Genetic cardiomyopathy is a group of intractable cardiovascular disorders involving heterogeneous genetic contribution. This heterogeneity has hindered the development of life-saving therapies for this serious disease. Genetic mutations in dystrophin and its associated glycoproteins cause cardiomuscular dysfunction. Large animal models incorporating these genetic defects are crucial for developing effective medical treatments, such as tissue regeneration and gene therapy. In the present study, we knocked out the δ-sarcoglycan (δ-SG) gene (SGCD) in domestic pig by using a combination of efficient de novo gene editing and somatic cell nuclear transfer. Loss of δ-SG expression in the SGCD knockout pigs caused a concomitant reduction in the levels of α-, ß-, and γ-SG in the cardiac and skeletal sarcolemma, resulting in systolic dysfunction, myocardial tissue degeneration, and sudden death. These animals exhibited symptoms resembling human genetic cardiomyopathy and are thus promising for use in preclinical studies of next-generation therapies.

The investigation of synergistic activity of protamine with conventional antimicrobial agents against oral bacteria.

In this study, we applied protamine, which is an antimicrobial peptide, to oral healthcare in combination with conventional antimicrobial agents. First, we explored the antimicrobial activity of protamine, with or without other antimicrobial agents, against Streptococcus mutans (S. mutans). Co-treatment with protamine and 3-methyl-4-isopropylphenol (IPMP) decreased the viability of S. mutans synergistically within 10 min. Interestingly, sodium fluoride (NaF) did not exhibit synergistic activity with protamine. Next, S. mutans and Streptococcus gordonii (S. gordonii) were co-treated with protamine and IPMP for 5 min to simulate tooth brushing. As a result, this co-treatment killed S. mutans faster than S. gordonii. Therefore, co-treatment with protamine and IPMP could be incorporated into oral healthcare products to prevent dental caries.

Fabrication of Gentamicin-Loaded Hydroxyapatite/Collagen Bone-Like Nanocomposite for Anti-Infection Bone Void Fillers.

A gentamicin-loaded hydroxyapatite/collagen bone-like nanocomposite (GNT-HAp/Col) was fabricated and evaluated for its absorption-desorption properties, antibacterial efficacy, and cytotoxicity. The hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) powder was mixed with gentamicin sulfate (GNT) in phosphate-buffered saline (PBS) at room temperature. After 6 h mixing, the GNT adsorption in all conditions reached plateau by Langmuir's isotherm, and maximum GNT adsorption amount was 34 ± 7 µg in 250 µg/mL GNT solution. Saturated GNT-loaded HAp/Col powder of 100 mg was soaked in 10 mL of PBS at 37 °C and released all GNT in 3 days. A shaking culture method for a GNT extraction from the GNT-HAp/Col and an inhibition zone assay for the GNT-HAp/Col compact showed antibacterial efficacy to Escherichia coli (E. coli) at least for 2 days. From the release profile of the GNT from the GNT-HAp/Col powder, antibacterial efficacy would affect E. coli at least for 3 days. Further, no cytotoxicities were observed on MG-63 cells. Thus, the GNT-HAp/Col is a good candidate of bioresorbable anti-infection bone void fillers by prevention initial infections, which is the primary cause of implant-associated infection even for rapid bioresorbable materials.

Antimicrobial Activity of Protamine-Loaded Calcium Phosphates against Oral Bacteria.

Protamine is an antimicrobial peptide extracted from fish. In this study, we loaded protamine onto dicalcium phosphate anhydride (DCPA), a dental material. Protamine was loaded by stirring DCPA into a protamine solution. To explore the antimicrobial activity of the materials, we cultivated Streptococcus mutans on fabricated discs for 24 h. When S. mutans was cultivated on the discs under no sucrose conditions, the loaded protamine was not released, and the ratio of dead bacteria increased on the surface of P (125) DCPA (half of the saturated level of protamine (125 ppm protamine) was loaded). Aside from P (500) DCPA (saturated level of protamine was loaded), some protamine was released, and the number of planktonic bacteria in the supernatant decreased. Using medium containing 1% sucrose, the release of protamine was promoted from P (125) DCPA due to lowered pH. However, lowering of the pH decreased the antimicrobial activity of protamine. On the other hand, P (500) DCPA released protamine before the pH was lowered, and biofilm formation was inhibited. The loaded protamine expressed antimicrobial activity, both on the surface of the materials and in the surrounding environment. The interaction of loaded protamine with calcium phosphates could promote the application of protamine in the dental field.

Co-Culture of Osteoblasts and Endothelial Cells on a Microfiber Scaffold to Construct Bone-Like Tissue with Vascular Networks.

Bone is based on an elaborate system of mineralization and vascularization. In hard tissue engineering, diverse biomaterials compatible with osteogenesis and angiogenesis have been developed. In the present study, to examine the processes of osteogenesis and angiogenesis, osteoblast-like MG-63 cells were co-cultured with human umbilical vein endothelial cells (HUVECs) on a microfiber scaffold. The percentage of adherent cells on the scaffold was more than 60% compared to the culture plate, regardless of the cell type and culture conditions. Cell viability under both monoculture and co-culture conditions was constantly sustained. During the culture periods, the cells were spread along the fibers and extended pseudopodium-like structures on the microfibers three-dimensionally. Compared to the monoculture results, the alkaline phosphatase activity of the co-culture increased 3-6 fold, whereas the vascular endothelial cell growth factor secretion significantly decreased. Immunofluorescent staining of CD31 showed that HUVECs were well spread along the fibers and formed microcapillary-structures. These results suggest that the activation of HUVECs by co-culture with MG-63 could enhance osteoblastic differentiation in the microfiber scaffold, which mimics the microenvironment of the extracellular matrix. This approach can be effective for the construction of tissue-engineered bone with vascular networks.

Acceleration of Osteogenesis via Stimulation of Angiogenesis by Combination with Scaffold and Connective Tissue Growth Factor.

In bone regeneration, there are some important cellular biological processes, such as mineralization, cell organization, and differentiation. In particular, vascularization into regenerative tissues is a key step for the survival of cells and tissues. In this study, to fabricate biomimetic-engineered bone, including vascular networks, we focused on connective tissue growth factor (CTGF), a multifunctional protein which could regulate the extracellular matrix remodeling. By combination with CTGF and hydroxyapatite (HAp) ceramics (2D) or apatite-fiber scaffold (AFS, 3D), we have fabricated bioactive materials. The CTGF-loaded HAp ceramics could enhance the cellular attachment through interaction with integrin and promote actin cytoskeletal reorganization. CTGF-loaded HAp also enhanced the differentiation of osteoblasts by integrin-mediated activation of the signaling pathway. Under co-culture conditions, both osteoblasts and endothelial cells in the CTGF-loaded AFS were stimulated by CTGF, and each cell could penetrate the central region of the scaffold in vitro and in vivo. Direct cell-cell interaction would also improve the functionality of cells in bone formation. These results suggest that coupling between effective optimized scaffold and CTGF with multifunction could provide better mimicking natural bone by stimulation of angiogenesis.

Distributions of endocrine cell clusters during porcine pancreatic development.

BACKGROUND: Pancreatic islet xenotransplantation is a potential treatment for diabetes mellitus, and porcine pancreas may provide a readily available source of islets. Islets in juvenile pigs are smaller than those in young adult pigs, but the insulin content is very similar. In addition, as juvenile pigs are more easily reared in uncontaminated conditions, many researchers have conducted studies using pancreatic islets from juvenile pigs. We aimed to analyze the distributions of endocrine cell clusters by comprehensively evaluating juvenile porcine pancreatic development and to propose an appropriate age at which islets could be isolated from the juvenile porcine pancreas. METHODS: Splenic (SL) and duodenal lobe (DL) samples were collected from the pancreases of pigs aged 0-180 days (n = 3/day after birth). The chronological changes in endocrine cell clustering were analyzed in relation to morphological changes, cell characterization, numbers, islet areas, and gene expression. RESULTS: In juvenile pigs aged 0-21 days, the pancreas contained numerous endocrine cells, and compact islets appeared from 21 days of age. Well-defined small islets were seen at 28 days of age, and the clusters were denser in the SL than in the DL. At 35 days of age, the islets were morphologically similar to those observed at 180 days of age, and the greater number of islets was similar to that seen at 90 days of age. The differences in the islets' cytoarchitecture between the lobes were negligible. The expression of ß-cell-related genes was higher in the juvenile pancreas than in the adult pancreas, and the expression of neurogenin-3 decreased dramatically over time. CONCLUSIONS: These findings may have implications for attempts to refine the most appropriate age for islet isolation from porcine donors. Focusing on porcine pancreatic islets isolated at around 35 days after birth may offer benefits regarding their xenotransplantation potential.

Fabrication of chelate-setting α-tricalcium phosphate cement using sodium citrate and sodium alginate as mixing solution and its in vivo osteoconductivity.

Moldable and injectable calcium-phosphate cements (CPCs) are material candidates for bone replacement applications. In the present study, we examined the effectiveness of sodium alginate and sodium citrate additives to the liquid phase of CPC, in improving its handling property as well as mechanical strength. The use of these additives enhanced the handling property significantly, in terms of consistency as compared to CPC without additives due to the liquefying effect caused by the adsorption of citrate ions on the cement particles. Sodium alginate and sodium citrate were added to CPC, which was set by the chelate-bonding capability of inositol phosphate, and was composed of mainly α-tricalcium phosphate (α-TCP) phase (>90%). The compressive strength of the CPC containing sodium alginate and sodium citrate was 3.4 ± 0.3 MPa, which was significantly higher than cement without additives. Furthermore, this cement exhibited favorable osteoconductivity and bioresorbability, and remained the α-TCP phase after 4-week implantation in a pig tibiae model. These results suggested that the cement is a potential candidate as a bioresorbable paste-like artificial bone. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2361-2370, 2018.

Effects of Adding Polysaccharides and Citric Acid into Sodium Dihydrogen Phosphate Mixing Solution on the Material Properties of Gelatin-Hybridized Calcium-Phosphate Cement.

We have succeeded in improving the material properties of a chelate-setting calcium-phosphate cement (CPC), which is composed of hydroxyapatite (HAp) the surface of which has been modified with inositol hexaphosphate (IP6) by adding α-tricalcium phosphate (α-TCP) powder. In order to create a novel chelate-setting CPC with sufficient bioresorbability, gelatin particles were added into the IP6-HAp/α-TCP cement system to modify the material properties. The effects of adding polysaccharides (chitosan, chondroitin sulfate, and sodium alginate) into the sodium dihydrogen phosphate mixing solution on the material properties of the gelatin-hybridized CPC were evaluated. The results of mechanical testing revealed that chondroitin sulfate would be the most suitable for fabricating the hybridized CPC with higher compressive strength. Moreover, further addition of an appropriate amount of citric acid could improve the anti-washout capability of the cement paste. In summary, a gelatin-hybridized IP6-HAp/α-TCP cement system prepared with a mixing solution containing chondroitin sulfate and citric acid is expected to be a beneficial CPC, with sufficient bioresorbability and material properties.