Erratum to: Milk Containing BF-7 Enhances the Learning and Memory, Attention, and Mathematical Ability of Normal Persons.

[This corrects the article DOI: 10.5851/kosfa.2009.29.2.278.].

Comparison of unprocessed silk cocoon and silk cocoon middle layer membranes for guided bone regeneration.

BACKGROUND: Silk cocoon is composed of multiple layers. The natural silk cocoon containing all layers was cut as a rectangular shape as defined as total group. The inner and outermost layers were removed from the total group and the remained mat was defined as the middle group. The objectives of this study was to compare the total group with the middle group as a barrier membrane for the guided bone regeneration. METHODS: The effects of these materials on the cellular proliferation and alkaline phosphatase (ALP) expression of MG63 cells were explored. For comparing bone regeneration ability, bilateral bone defects were created in calvarial areas in ten adult New Zealand white rabbits. The defects were covered with silk membranes of the middle group, with silk membrane of the total group used as the control on the contralateral side. The defects were allowed to heal for 4 and 8 weeks. Micro-computerized tomography (µCT) and histological examination were performed. RESULTS: The middle group exhibited a higher MTT value 48 and 72 h after treatment compared to the total group. ALP expression was also higher in the middle group. The results of µCT and histologic examination showed that new bone formation was significantly higher in the middle group compared to the total group 8 weeks postoperatively (P < 0.05). CONCLUSIONS: In conclusion, the middle layer of the silk cocoon supports guided bone regeneration better than unprocessed silk cocoon.

Novel fabrication of fluorescent silk utilized in biotechnological and medical applications.

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine.

Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway.

The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca(2+)-activated K(+) channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21(Cip1) in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats.

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10-mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p<0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p<0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p<0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury.

Inhibitory effects of bee venom on Propionibacterium acnes-induced inflammatory skin disease in an animal model.

Propionibacterium acnes (P. acnes) is a major contributing factor to the inflammatory component of acne. The many prescription medications for acne allow for a large number of potential combination treatments. However, several antibiotics, apart from their antibacterial effects, exert side­effects, such as the suppression of host inflammatory responses. Purified bee venom (BV) is a natural toxin produced by honeybees (Apis mellifera L.). BV has been widely used as a traditional medicine for various diseases. In the present study, to investigate the therapeutic effects of BV against P. acnes-induced inflammatory skin disease, P. acnes was intradermally injected into the ears of mice. After the injection, BV was applied to the skin surface of the right ear. Histological observation revealed that P. acnes induced a considerable increase in the number of infiltrated inflammatory cells. However, treatment with BV markedly reduced these reactions compared with the P. acnes-injected mice not treated with BV. Moreover, the expression levels of tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß were significantly reduced in the BV-treated mice compared with the untreated P. acnes-injected mice. In addition, treatment with BV significantly inhibited Toll-like receptor (TLR)2 and CD14 expression in P. acnes-injected tissue. The binding activity of nuclear factor-κB (NF-κB) and activator protein (AP)-1 was markedly suppressed following treatment with BV. The results from our study, using an animal model, indicate that BV exerts an inhibitory effect on inflammatory skin diseases. In conclusion, our data indicate that BV has potential for use as an anti-acne agent and may be useful in the pharmaceutical and cosmetics industries.

Powdered Wound Dressing Materials Made from wild Silkworm Antheraea pernyi Silk Fibroin on Full-skin Thickness Burn Wounds on Rats.

PURPOSE: This study evaluated powdered burn wound dressing materials from wild silkworm fibroin in an animal model. METHODS: Fifteen rats were used in this experiment. Full-thickness 2×2 cm burn wounds were created on the back of rats under anesthesia. In the two experimental groups, the wounds were treated with two different dressing materials made from silkworm fibroin. In the Control Group, natural healing without any dressing material was set as control. The wound surface area was measured at five days, seven days and 14 days. Wound healing was evaluated by histologic analysis. RESULTS: By gross observation, there were no infections or severe inflammations through 14 days post-injury. The differences among groups were statistically significant at seven days and 14 days, postoperatively (P <0.037 and 0.001, respectively). By post hoc test, the defect size was significantly smaller in experimental Group 1 compared with the Control Group and experimental Group 2 at seven days postoperatively (P =0.022 and 0.029, respectively). The difference between Group 1 and Group 2 was statistically significant at 14 days postoperatively (P <0.001). Group 1 and control also differed significantly (P =0.002). Group 1 showed a smaller residual scar than the Control Group and Group 2 at 14 days post-injury. Histologic analysis showed more re-epithelization in Groups 1 and 2 than in the Control Groups. CONCLUSION: Burn wound healing was accelerated with silk fibroin spun by wild silkworm Antheraea pernyi. There was no atypical inflammation with silk dressing materials. In conclusion, silk dressing materials can be used for treatment of burn wound.

Bee venom ameliorates compound 48/80-induced atopic dermatitis-related symptoms.

UNLABELLED: Honeybee (Apis mellifera L.) venom (BV) has been traditionally used for the treatment of pain and inflammatory diseases such as itchy skin problems. However, the precise mechanism of BV in ameliorating the scratching behavior is not fully understood. OBJECTIVE: In order to evaluate the effect of BV on atopic dermatitis-related symptoms in mice, we used a mouse skin scratching model induced by compound 48/80. The anti-itch effect of BV was investigated in a compound 48/80-induced mouse scratching behavior model. BALB/c mice were injected intraperitoneally with vehicle (saline 0.9%) or BV (0.01 and 0.1 mg/kg). One hour after treatment, the animals received a subcutaneous injection of compound 48/80. Intraperitoneal administration of BV (0.01 and 0.1 mg/kg) attenuated compound 48/80-induced scratching behaviors. The anti-scratching behavior effect of BV was in proportional to its vascular permeability effects. Treatment with BV also inhibited the degranulation of mast cells and the production of pro-inflammatory cytokines in compound 48/80-treated skin tissues. According to these results, BV may improve atopic dermatitis-related symptoms by inhibiting the mast cell degranulation and pro-inflammatory cytokine expression.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

The objective of this study was to demonstrate that a silk fibroin (SF) and 4-hexylresorcinol (4-HR) incorporation membrane could be used for a guided bone regeneration technique. Fourier transform infrared measurements were obtained to determine change of physical property of SF membrane by 4-HR incorporation. Two peri-implant defects, 3.0 × 5.0 mm (width × length), were prepared on the lateral side of the implant hole in the tibia of New Zealand white rabbits (n = 8). The peri-implant defect was left unfilled in the control group. Silk fibroin + 4-HR membrane was applied to the peri-implant defect in the experimental group. The 8 animals were killed at 8 weeks after implantation. Subsequently, removal torque test and histomorphometric evaluation were done. Fourier transform infrared spectroscopy showed no specific chemical interaction between 4-HR and SF. In the histomorphometric analysis, the mean bone regeneration was 18.3 ± 1.9 mm(2) in the experimental group and 9.3 ± 0.9 mm(2) in the control group (P = 0.004). In conclusion, the SF and 4-HR incorporation membrane successfully regenerated bone in the rabbit tibia peri-implant bone defect model.

Effects of cosmetics containing purified honeybee (Apis mellifera L.) venom on acne vulgaris.

OBJECTIVE: Acne vulgaris is a chronic dermatologic problem with multiple factors involved in its pathogenesis. Alternative solutions to acne treatment were instigated by antibiotic resistance despite of its extensive use. Purified bee venom (PBV) has been proposed as a promising candidate for that purpose. The present study was designed to confirm the antibacterial effect of PBV and access the efficacy of cosmetics containing PBV in subjects with acne vulgaris. METHODS: The skin bacterium Propionibacterium acnes was incubated with PBV at various concentrations and bacterial growth was evaluated using the colony forming unit (CFU) assay. The mechanism of PBV employed in killing P. acnes was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, a total of 12 subjects were randomized in a double-blind, controlled trial to receive either cosmetics containing PBV or cosmetics without PBV for two weeks. Evaluations included lesion counts and skin microorganism. RESULTS: PBV exhibited antimicrobial activity in a concentration-dependent manner, reducing the number of P. acnes CFU by approximately 6 logs at a concentration of 0.5 mg. When PBV concentration was higher than 1.0 mg, no P. acnes colonies were spotted on an agar. TEM and SEM of untreated P. acnes illustrated the normal pleomorphic structure, whereas the PBV-treated bacterium lost the integrity of surface architecture. Significant difference (P=0.027) in the grading levels based on numbers of lesion counts for inflammatory and noninflammatory was observed in favour of the PBV group compared with the control group. In terms of average decrement of skin microorganism, subjects receiving cosmetics containing PBV experienced a significant 57.5% decrease of adenosine triphosphate levels, whereas participants receiving cosmetics without PBV experienced a nonsignificant decrease of 4.7%. CONCLUSION: These results show that the in vitro actions of antimicrobial activity of PBV were translated in vivo. Cosmetics containing PBV provided a certain degree of efficacy in terms of lesion counts and skin microorganism concentration compared with cosmetics without PBV in subjects with acne vulgaris. PBV may be a good candidate compound for developing therapeutic drug for the treatment of acne vulgaris.

Skin sensitization study of bee venom (Apis mellifera L.) in guinea pigs and rats.

BACKGROUND: Bee venom (Apis mellifera L., BV) possessing a rich source of pharmacologically active substances has the potential to be used as a cosmetic ingredient for antiaging, antiinflammatory and antibacterial functions. The aim of this study was to assess the skin sensitization of BV on experimental animals using the Buehler test. MATERIALS AND METHODS: Guinea pigs were randomly allocated into three groups of BV-sensitization, positive control-sensitization, and ethyl alcohol-sensitization group for induction and challenge. On the other hand, two groups of rats were administered with BV at doses of 0 and 1500 mg/kg. Clinical signs, mortality and body weight changes were continually monitored during the study period. RESULTS: No treatment-related clinical signs or body weight changes were observed in both animal models. The average skin reaction evaluated by erythema and edema on the challenge sites, and sensitization rate in the BV-sensitization group of guinea pigs were substantially low compared with those in positive control group, representing a negligible sensitizing potential of BV. CONCLUSION: It was concluded that BV was well tolerated and exhibited no dermal irritation potential in guinea pigs and rats. Our findings may provide a developmental basis of BV for a cosmetic ingredient or external application for topical uses.

Silk fibroin hydrolysate inhibits osteoclastogenesis and induces apoptosis of osteoclasts derived from RAW 264.7 cells.

Bone disease can be associated with bone resorption by osteoclasts, and interest in the development of antiresorptive agents has recently increased. The hydrolysate of silk fibroin has been studied with respect to such biomedical applications. In a previous study, silk fibroin showed indirect inhibitory effects on the differentiation of osteoclasts. To further evaluate the effect of a hydrolysate of silk fibroin on osteoclasts, we investigated the direct effects of the silk fibroin hydrolysate on osteoclastogenesis and apoptosis of osteoclasts induced by receptor activation of nuclear factor κB ligand (RANKL). The silk fibroin hydrolysate inhibited RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP) in RAW 264.7 cells. The inhibitory effect of the silk fibroin hydrolysate resulted in the decreased expression of osteoclast marker genes, such as matrix metalloproteinase-9 (MMP-9), cathepsin-K and calcitonin receptor (CTR). In addition, the silk fibroin hydrolysate blocked the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and expression of transcription factors, such as nuclear factor of activated T cells c1 (NFATc1) and NF-κB. Finally, the silk fibroin hydrolysate induced apoptosis signaling cascades. Taken together, the present results indicate that silk fibroin hydrolysate has antiresorptive activity by both inhibiting osteoclastogenesis and inducing osteoclast apoptosis.

Dermal and ocular irritation studies of honeybee (Apis mellifera L.) venom.

The aim of this study was to access the irritant properties of bee venom (BV) after its application to skin and eye mucous membranes of the rabbit. The animals were also observed for clinical signs and mortality after the application of the test material. Six animals were used for the skin irritation test and nine rabbits for the eye irritation test. The acute BV application to the rabbit skin revealed no appreciable clinical signs throughout the observation period of 72 h and there was no mortality seen. In the eye irritation test, eye reactions were read and graded 24, 48, 72, 96 and 168 h after BV treatment. No changes in the cornea, iris or conjunctivae were observed at all time points of observations. Based on the present findings, it can be concluded that the irritation potential of BV is negligible.

Enhancement of anti-inflammatory activity of PEP-1-FK506 binding protein by silk fibroin peptide.

Silk fibroin (SF) peptide has been traditionally used as a treatment for flatulence, spasms, and phlegm. In this study, we examined whether SF peptide enhanced the antiinflammatory effect of PEP-1-FK506 binding protein (PEP-1-FK506BP) through comparing the anti-inflammatory activities of SF peptide and/or PEP-1-FK506BP. In the presence or absence of SF peptide, transduction levels of PEP-1-FK506BP into HaCaT cells and mice skin and anti-inflammatory activities of PEP-1-FK506BP were identified by Western blot and histological analyses. SF peptide alone effectively reduced both mice ear edema and the elevated levels of cyclooxygenase-2, interleukin-6 and -1beta, and tumor necrosis factor-alpha, showing similar anti-inflammatory effect to that of PEP-1-FK506BP. Furthermore, co-treatment with SF peptide and PEP-1- FK506BP exhibited more enhanced anti-inflammatory effects than the samples treated with SF peptides or PEP- 1-FK506BP alone, suggesting the possibility that SF peptide and PEP-1-FK506BP might interact with each other. Moreover, the transduction data demonstrated that SF peptide did not affect the transduction of PEP-1- FK506BP into HaCaT cells and mice skin, indicating that the improvement of anti-inflammatory effect of PEP-1- FK506BP was not caused by enhanced transduction of PEP-1-FK506BP. Thus, these results suggest the possibility that co-treatment with SF peptide and PEP-1-FK506BP may be exploited as a useful therapy for various inflammationrelated diseases.

Effect of silk fibroin peptide derived from silkworm Bombyx mori on the anti-inflammatory effect of Tat-SOD in a mice edema model.

We investigated whether silk fibroin peptide derived from the silkworm, Bombyx mori, could inhibit inflammation and enhance the anti-inflammatory activity of Tat-superoxide dismutase (Tat-SOD), which was previously reported to effectively penetrate various cells and tissues and exert anti-oxidative activity in a mouse model of inflammation. Inflammation was induced by topical treatment of mouse ears with 12-O-tetradecanoylphorbol-13-acetate (TPA). Histological, Western blot, and reverse transcription-polymerase chain reaction data demonstrated that silk fibroin peptide or Tat-SOD alone could suppress elevated levels of cyclooxygenase-2, interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha induced by TPA. Moreover, silk fibroin peptide significantly enhanced the anti-inflammatory activity of Tat-SOD, although it had no influence on in vitro and in vivo transduction of Tat-SOD. Silk fibroin peptide exhibited anti- inflammatory activity in a mice model of inflammation. Therefore, silk fibroin peptide alone or in combination with Tat-SOD might be used as a therapeutic agent for various inflammatory diseases.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

Silk peptides inhibit adipocyte differentiation through modulation of the Notch pathway in C3H10T1/2 cells.

Silk protein is a biocompatible material that has been used in many biotechnological applications and exhibits body fat-lowering effects. Recent studies have shown that silk peptides increase expression of osteogenic markers in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are inverse processes and often regulated reciprocally, we hypothesized that silk peptides might suppress adipocyte differentiation. We therefore endeavored to evaluate the effects of silk peptides on adipocyte differentiation in C3H10T1/2 cells. We find that silk peptides inhibit lipid accumulation and morphological differentiation in these cells. Molecular studies show that silk peptides block expression of adipocyte-specific genes such as peroxisome proliferator-activated receptor γ and its targets, including aP2, Cd36, CCAAT enhancer binding proteinα. Silk peptides appear to inhibit adipogenesis by suppression of the Notch pathway, repressing the Notch target genes Hes-1 and Hey-1. In addition, these peptides inhibit endogenous Notch activation, as shown by a reduction in generation of Notch intracellular domain. N-[N-(3.5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester, compound E, and WPE-III-31C, which are all known Notch signaling inhibitors, block adipocyte differentiation to an extent similar to silk peptides. Together, our data demonstrate that silk peptides can modulate adipocyte differentiation through inhibition of the Notch signaling and further suggest potential future strategies for treating obesity and its related metabolic diseases.

Royal jelly protects against ultraviolet B-induced photoaging in human skin fibroblasts via enhancing collagen production.

Royal jelly (RJ) is a honeybee product containing proteins, carbohydrates, fats, free amino acids, vitamins, and minerals. As its principal unsaturated fatty acid, RJ contains 10-hydroxy-2-decenoic acid (10-HDA), which may have antitumor and antibacterial activity and a capacity to stimulate collagen production. RJ has attracted interest in various parts of the world for its pharmacological properties. However, the effects of RJ on ultraviolet (UV)-induced photoaging of the skin have not been reported. In this study we measured the 10-HDA content of RJ by high-performance liquid chromatography and tested the effects of RJ on UVB-induced skin photoaging in normal human dermal fibroblasts. The effects of RJ and 10-HDA on UVB-induced photoaging were tested by measuring procollagen type I, transforming growth factor (TGF)-ß1, and matrix metalloproteinase (MMP)-1 after UVB irradiation. The RJ contained about 0.211% 10-HDA. The UVB-irradiated human skin fibroblasts treated with RJ and 10-HDA had increased procollagen type I and TGF-ß1 productions, but the level of MMP-1 was not changed. Thus RJ may potentially protect the skin from UVB-induced photoaging by enhancing collagen production.

Accelerated healing with the use of a silk fibroin membrane for the guided bone regeneration technique.

OBJECTIVE: The purpose of this study was to evaluate the bone regeneration ability of silk fibroin (SF) membrane. STUDY DESIGN: Fourier-transform infrared (FT-IR) and solubility test against distilled water were performed with 3 different types of SF membrane (SM1, SM2, and SM3). Subsequently, microscopic computerized tomography (µ-CT) and histomorphometric analyses were performed in rabbit calvarial defect model after SF membrane application at 4 and 8 weeks after surgery. RESULTS: FT-IR showed that the conformation of the SF membrane was a random coil structure and that SM1 was the least soluble. When SM1 was used in the animal model, the groups with SM1 had significantly higher new bone formation than the uncovered control in both the µ-CT and the histomorphometric analyses (P < .05). CONCLUSIONS: The SF membrane had more new bone formation compared with the uncovered control.

Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new bone substitute.

PURPOSE: This study involves a comparison between the bone regeneration of nano-hydroxyapatite (nHA), as derived from eggshells either with or without silk fibroin scaffolds, and the unfilled control in the rabbit calvarial bony defect model. MATERIALS AND METHODS: Sixteen 4-month-old New Zealand white rabbits, with a mean weight of 2.8 kg (range, 2.5-3.0 kg), were used in this experiment. After the formation of bilateral parietal bony defects (diameter, 8.0 mm), either an nHA or an nHA+silk fibroin combination (nHA+silk) was grafted. The control was unfilled defect. The bone regeneration was evaluated by micro-computed tomography (µCT) and histomorphometric analyses at 4 and 8 weeks. RESULTS: All measured variables of the µCT analysis were significantly higher in the grafted groups (nHA and nHA+silk) than in the unfilled control groups at both 4 and 8 weeks after operation (P < .05). On histomorphometric analysis, there was no significant difference between the groups at 4 weeks after operation. However, the nHA group exerted significantly higher bone regeneration (40.16% ± 8.27%) compared with the unfilled control group (25.66% ± 10.98%) or the nHA+silk group (16.62% ± 3.05%) (P < .05). CONCLUSION: The nHA from eggshells exerted better bone formation than the unfilled control group on both µCT and histomorphometric analyses. Considering the rapid healing in bony defect and easy availability, the nHA from the eggshells could prove to be a good new bone substitute.