Repair of critical-sized bone defects in rabbit femurs using graphitic carbon nitride (g-C3N4) and graphene oxide (GO) nanomaterials.

Various biomaterials have been evaluated to enhance bone formation in critical-sized bone defects; however, the ideal scaffold is still missing. The objective of this study was to investigate the in vitro and in vivo regenerative capacity of graphitic carbon nitride (g-C3N4) and graphene oxide (GO) nanomaterials to stimulate critical-sized bone defect regeneration. The in vitro cytotoxicity and hemocompatibility of g-C3N4 and GO were evaluated, and their potential to induce the in vitro osteogenesis of human fetal osteoblast (hFOB) cells was assessed using qPCR. Then, bone defect in femoral condyles was created in rabbits and left empty as control or filled with either g-C3N4 or GO. The osteogenesis of the different implanted scaffolds was evaluated after 4, 8, and 12 weeks of surgery using X-ray, computed tomography (CT), macro/microscopic examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions. Both materials displayed good cell viability and hemocompatibility with enhanced collagen type-I (Col-I), OC, and OP expressions of the hFOB cells. Compared to the control group, the bone healing process in g-C3N4 and GO groups was promoted in vivo. Moreover, complete healing of the bone defect was observed radiologically and grossly in g-C3N4 implanted group. Additionally, g-C3N4 implanted group showed higher percentages of osteoid tissue, mature collagen, biodegradation, and expressions of OC and OP. In conclusion, our results revealed that g-C3N4 and GO nanomaterials could induce osteogenesis in critical-sized bone defects.

Assessment of hemodynamics, blood gases, and lung histopathology of healthy Pig model on two different mechanical ventilators.

In response to COVID-19 global crisis and arising from social responsibility, efforts have been exerted to promptly research, develop and manufacture ICU ventilators locally to meet the spike in demand. This study aimed at : Evaluating the safety and performance of a newly developed mechanical ventilator; EZVent compared to a commercial ventilator regarding hemodynamics, arterial blood gases (ABG), lung inflammatory markers, and histopathology in a healthy pig model using three different ventilation modes. Methods: Eight adult male pigs were anesthetized and randomly assigned into two equal groups: Commercial vent and EZVent group, the animals of which were ventilated using a standard commercial ventilator and EZVent, respectively. On every animal, three ventilation modes were tested, each mode for 30 min: CMV-VC, CMV-PC, and CPAP-PS modes. Vital signs, ECG, Lung Mechanics (LM), and ABG were measured before ventilation and after 30 min of ventilation of each mode. After animals' euthanasia, histological examinations of lung samples including morphometric assessment of alveolar edema, alveolar wall thickening, and the mean number of inflammatory cellular infiltrate/cm2 of lung tissue were analyzed. TNF-α and Il-6 expression and localization in lung tissue were assessed by western blot and immunohistochemistry. Results: The vital signs, LM, ABG, morphometric analysis, and histopathological score during the different ventilation modes showed non-significant differences between the study groups. TNF-α and IL-6 were minimally expressed in the bronchiolar epithelium and the alveolar septa. Their increased expression level was insignificant. Conclusion: EZVent is equivalent to the commercial ventilator regarding its safety and efficacy.

Enhancement of critical-sized bone defect regeneration using UiO-66 nanomaterial in rabbit femurs.

BACKGROUND: Repair of large-sized bone defects is a challengeable obstacle in orthopedics and evoked the demand for the development of biomaterials that could induce bone repair in such defects. Recently, UiO-66 has emerged as an attractive metal-organic framework (MOF) nanostructure that is incorporated in biomedical applications due to its biocompatibility, porosity, and stability. In addition, its osteogenic properties have earned a great interest as a promising field of research. Thus, the UiO-66 was prepared in this study and assessed for its potential to stimulate and support osteogenesis in vitro and in vivo in a rabbit femoral condyle defect model. The nanomaterial was fabricated and characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM). Afterward, in vitro cytotoxicity and hemolysis assays were performed to investigate UiO-66 biocompatibility. Furthermore, the material in vitro capability to upregulate osteoblast marker genes was assessed using qPCR. Next, the in vivo new bone formation potential of the UiO-66 nanomaterial was evaluated after induction of bone defects in rabbit femoral condyles. These defects were left empty or filled with UiO-66 nanomaterial and monitored at weeks 4, 8, and 12 after bone defect induction using x-ray, computed tomography (CT), histological examinations, and qPCR analysis of osteocalcin (OC) and osteopontin (OP) expressions. RESULTS: The designed UiO-66 nanomaterial showed excellent cytocompatibility and hemocompatibility and stimulated the in vitro osteoblast functions. The in vivo osteogenesis was enhanced in the UiO-66 treated group compared to the control group, whereas evidence of healing of the treated bone defects was observed grossly and histologically. Interestingly, UiO-66 implanted defects displayed a significant osteoid tissue and collagen deposition compared to control defects. Moreover, the UiO-66 nanomaterial demonstrated the potential to upregulate OC and OP in vivo. CONCLUSIONS: The UiO-66 nanomaterial implantation possesses a stimulatory impact on the healing process of critical-sized bone defects indicating that UiO-66 is a promising biomaterial for application in bone tissue engineering.

In vitro and in vivo Effects of a Single Dose of Bupivacaine 5% on Donkey Chondrocytes.

Single intra-articular (IA) injection of long-acting local anesthetics such as bupivacaine is commonly used clinically for postoperative analgesia, in particular, after arthroscopic surgery. Despite their widespread use, the side effects of IA bupivacaine on joint cartilage as well as hepatotoxic and nephrotoxic effects remain to be elucidated. The aim of this study is to assess the in vitro effect of bupivacaine 5% on donkey chondrocytes at different time points, in addition to the in vivo effects of a single IA bupivacaine injection on the middle carpal joint in a group of 10 clinically healthy adult male donkeys. In phase I, the effect of in vitro treatment with bupivacaine 5% or saline 0.9% on freshly isolated donkey chondrocytes for 30, 60 min, 24, 48, and 96 h was investigated using MTT and LIVE/DEAD assay. In phase II, in vivo effects of single injection of bupivacaine on the middle carpal joint of the donkey were evaluated compared with saline 0.9%. Biochemical analysis of collected serum and synovia was performed. Additionally, articular cartilage damage was evaluated using radiography, computed tomography (CT), catabolic marker expression via quantitative polymerase chain reaction (qPCR), and histopathological examination 96 h after injection. Our results showed that after a 30-min exposure to bupivacaine 5%, the viability of donkey chondrocytes was 97.3 ± 4.4% and was not significantly affected at the indicated time points (n = 8, p < 0.05). No significant changes in biochemical analytes of serum and synovial fluid following IA bupivacaine injection were observed, compared with saline injection (n = 5 for each group, p < 0.05). Furthermore, in vivo IA injection of bupivacaine revealed no significant differences in radiography, CT scan, gene expression of cartilage catabolic biomarkers, and histopathological examination. These results provide an evidence for the safety of bupivacaine on the donkey cartilage.

Effect of Topical Cyclopentolate 1% on Ocular Ultrasonographic Features, Intraocular Pressure, Tear Production, and Pupil Size in Normal Donkeys (Equus Asinus).

This study was performed to investigate the effects of cyclopentolate on ultrasonographic parameters of eye structures, intraocular pressure (IOP), tear production, and pupil size in normal donkeys. Sixteen eyes of eight clinically healthy adult donkeys (2-2.5 years old) weighing 295 ± 34 kg (mean ± standard deviation) were used in this study. Cyclopentolate hydrochloride 1% was instilled in a randomly selected eye and the other eye received normal saline drops as a control. The effect of cyclopentolate was evaluated by ultrasonography. Additionally, changes in IOP and tear production were evaluated for 2 hours post-instillation by tonometry and Schirmer tear test (STT), respectively. Vertical and horizontal pupil diameters were recorded pre-instillation (0), and 15, 30-, 45-, 60-, and 120-minutes post-instillation. After cyclopentolate 1% instillation, iridocorneal angle and width of the entry of ciliary cleft were significantly increased as observed by ultrasonography. IOP was significantly increased starting from 30 minutes till 60 minutes post-instillation of cyclopentolate 1%. Non-significant alteration in the STT was observed in the cyclopentolate-treated eyes compared to the control eyes. Both vertical and horizontal pupil diameters began to significantly increase 30 minutes after cyclopentolate 1% instillation compared to the control saline group. In conclusion, cyclopentolate 1% could be used as a potent cycloplegic drug in donkeys without systemic or ocular side effects.

Graphene oxide-cellulose nanocomposite accelerates skin wound healing.

The usage of materials with the potential to accelerate wound healing is a great benefit for patients and health care systems. This study evaluated the impact of using graphene oxide (GO)-cellulose nanocomposite on skin wound healing via in vitro and in vivo investigations. The nanomaterial was synthesized and characterized. Cytocompatibility performance of the GO-cellulose was investigated through in vitro testing based on MTT and live/dead assays by EA.hy926 human endothelial cells (ECs). Additionally, the effect of GO-cellulose on induced wound scratch model using EA.hy926 ECs was investigated. Finally, the therapeutic effect of GO-cellulose was evaluated in vivo after the creation of two full-thickness wounds in the dorsum of rats (8 mm diameter). These wounds were randomly placed into two groups, the control group (10 wounds) and the GO-cellulose group (10 wounds), and monitored for gross and histopathological changes at 7 and 21 days after wound induction. MTT and Live/Dead assays showed excellent GO-cellulose cytocompatibility, whereas no difference in ECs viability was observed after culturing using conditioned media. GO-cellulose nanocomposite enhanced cell migration in the in vitro wound scratch assay. As compared to the control group, the GO-cellulose nanocomposite group's wound healing process was promoted in the in vivo rat skin wounds. Interestingly, wound re-epithelization and neovascularization were significantly accelerated in the GO-cellulose-treated rats. Furthermore, thick granulation tissue formation and intense collagen deposition were found in the GO-cellulose group. These findings showed that GO-cellulose has a promoting effect on skin wound healing, suggesting its promising and potential application in tissue regeneration.

Development and Characterization of Cellulose/Iron Acetate Nanofibers for Bone Tissue Engineering Applications.

In tissue engineering, design of biomaterial with a micro/nano structure is an essential step to mimic extracellular matrix (ECM) and to enhance biomineralization as well as cell biocompatibility. Composite polymeric nanofiber with iron particles/ions has an important role in biomineralization and collagen synthesis for bone tissue engineering. Herein, we report development of polymeric cellulose acetate (CA) nanofibers (17 wt.%) and traces of iron acetates salt (0.5 wt.%) within a polymeric solution to form electrospinning nanofibers mats with iron nanoparticles for bone tissue engineering applications. The resulting mats were characterized using field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The resulted morphology indicated that the average diameter of CA decreased after addition of iron from (395 ± 30) to (266 ± 19) nm and had dense fiber distributions that match those of native ECM. Moreover, addition of iron acetate to CA solution resulted in mats that are thermally stable. The initial decomposition temperature was 300 °C of CA/Fe mat > 270 °C of pure CA. Furthermore, a superior apatite formation resulted in a biomineralization test after 3 days of immersion in stimulated environmental condition. In vitro cell culture experiments demonstrated that the CA/Fe mat was biocompatible to human fetal-osteoblast cells (hFOB) with the ability to support the cell attachment and proliferation. These findings suggest that doping traces of iron acetate has a promising role in composite mats designed for bone tissue engineering as simple and economically nanoscale materials. Furthermore, these biomaterials can be used in a potential future application such as drug delivery, cancer treatment, and antibacterial materials.

Small diameter polycaprolactone vascular grafts are patent in sheep carotid bypass but require antithrombotic therapy.

Background: Polycaprolactone (PCL) scaffolds exhibit high biocompatibility and are attractive as vascular conduits. Materials & methods: PCL tubes were cultivated in bioreactor with human adipose regenerative cells to assess ex vivo cytocompatibility, whereas in vivo PCL tube patency was evaluated in sheep carotid bypass with and without antithrombotic treatment. Results:Ex vivo results revealed increasing adipose regenerative cells on PCL using dynamic bioreactor culturing. In vivo data showed that 67% (2/3) of grafts in the antithrombotic group were patent at day 28, while 100% (3/3) of control grafts were occluded already during the first week due to thrombosis. Histology showed that patent PCL grafts were recellularized by host cells. Conclusion: PCL tubes may work as small diameter vascular scaffolds under antithrombotic treatment.

Custom-made artificial eyes using 3D printing for dogs: A preliminary study.

Various incurable eye diseases in companion animals often result in phthisis bulbi and eye removal surgery. Currently, the evisceration method using silicone balls is useful in animals; however, it is not available to those with impaired cornea or severe ocular atrophy. Moreover, ocular implant and prostheses are not widely used because of the diversity in animal size and eye shape, and high manufacturing cost. Here, we produced low-cost and customized artificial eyes, including implant and prosthesis, using computer-aided design and three-dimensional (3D) printing technique. For 3D modeling, the size of the artificial eyes was optimized using B-mode ultrasonography. The design was exported to STL files, and then printed using polycaprolactone (PCL) for prosthesis and mixture of PCL and hydroxyapatite (HA) for ocular implant. The 3D printed artificial eyes could be produced in less than one and half hour. The prosthesis was painted using oil colors and biocompatible resin. Two types of eye removal surgery, including evisceration and enucleation, were performed using two beagle dogs, as a preliminary study. After the surgery, the dogs were clinically evaluated for 6 months and then histopathological evaluation of the implant was done. Ocular implant was biocompatible and host tissue ingrowth was induced after in vivo application. The custom-made prosthesis was cosmetically excellent. Although long-term clinical follow-up might be required, the use of 3D printed-customized artificial eyes may be beneficial for animals that need personalized artificial eye surgery.

Decellularized hepatic extracellular matrix hydrogel attenuates hepatic stellate cell activation and liver fibrosis.

Liver fibrosis results from excessive accumulation of extracellular matrix (ECM) proteins that distort the hepatic architecture. Progression of liver fibrosis results in cirrhosis and liver failure, and often, liver transplantation is required. The decellularized liver tissue contains different components that mimic the natural hepatic environment. We hypothesized that a decellularized liver hydrogel can be used to replace the necrotic hepatocytes and damaged ECM. Therefore, our aim in this study is to develop a therapy for treating liver fibrosis. Mice livers were decellularized and processed to form a hepatic hydrogel. We evaluated the biocompatibility and bioactivity of the hydrogel. The ability of the hydrogel to enhance the migration of hepatocytes and endothelial cells was investigated. Human hepatic stellate cell line (LX-2) activated by transforming growth factor-ß1 (TGF-ß1) was used as in vitro model for fibrogenesis. Then, the hydrogel was injected into the liver parenchyma of mice after the induction of liver fibrosis using thioacetamide. The resulting hydrogel maintained a complex composition, which included glycosaminoglycans, collagen, elastin, and growth factors. Hepatocytes and endothelial cells were shown to migrate toward the hydrogel in vitro. Liver hydrogel improved TGF-ß1-induced LX-2 cells activation via blocking the TGF-ß1/Smad pathway. The matrix was delivered successfully in vivo and enhanced the reduction of fibrosis and recovery to a nearly normal structure. In conclusion, we have demonstrated that the liver hydrogel can be utilized as an injectable biomaterial for liver tissue engineering in order to reduce the degree of fibrosis.

Congenital urethral dilatation in cattle calves: Diagnosis and surgical intervention.

BACKGROUND AND AIM: Congenital anomalies of the urinary system are common affections in ruminants. Dilatation of the pelvic urethra is one of these affections in which the pelvic urethra dilated than normal diameter. This study aimed to explain the diagnosis and surgical treatment of urethral dilatation in cattle calves. MATERIALS AND METHODS: Twenty-three bull calves (2-7 months old) were presented with a history of stranguria, tenesmus, and straining. Diagnosis of urethral dilatation was relied on the case history and clinical examination and was confirmed using survey and contrast radiography, ultrasonography, and biochemical tests. Treatment was done by urethrostomy under the effect of local infiltration analgesia. RESULTS: Physical examination revealed the presence of an oval, firm, and painless swelling at the perineal region, starting just below the anus and extended to the base of the scrotum. The owners reported that the initial swelling size and severity of symptoms increased with the progress of animal age. Biochemical findings revealed non-significant changes in blood urea nitrogen and creatinine levels. Radiographic findings showed an oval radiopaque mass. However, a well-demarcated structure with acoustic enhancement was detected on ultrasonographic examination. Urethrostomy resulted in a successful outcome of all cases. CONCLUSION: Depending on these findings, ultrasonography is the most reliable diagnostic tool and urethrostomy is the intervention of choice with acceptable results for diagnosis and treatment of urethral dilatation in cattle calves, respectively.

Vascular reconstruction: A major challenge in developing a functional whole solid organ graft from decellularized organs.

Bioengineering a functional organ holds great potential to overcome the current gap between the organ need and shortage of available organs. Whole organ decellularization allows the removal of cells from large-scale organs, leaving behind extracellular matrices containing different growth factors, structural proteins, and a vascular network with a bare surface. Successful application of decellularized tissues as transplantable organs is hampered by the inability to completely reline the vasculature by endothelial cells (ECs), leading to blood coagulation, loss of vascular patency, and subsequent death of reseeded cells. Therefore, an intact, continuous layer of endothelium is essential to maintain proper functioning of the vascular system, which includes the transfer of nutrients to surrounding tissues and protecting other types of cells from shear stress. Here, we aimed to summarize the available cell sources that can be used for reendothelialization in addition to different trials performed by researchers to reconstruct vascularization of decellularized solid organs. Additionally, different techniques for enhancing reendothelialization and the methods used for evaluating reendothelialization efficiency along with the future prospective applications of this field are discussed. STATEMENT OF SIGNIFICANCE: Despite the great progress in whole organ decellularization, reconstruction of vasculature within the engineered constructs is still a major roadblock. Reconstructed endothelium acts as a multifunctional barrier of vessels, which can reduce thrombosis and help delivering of oxygen and nutrients throughout the whole organ. Successful reendothelialization can be achieved through reseeding of appropriate cell types on the naked vasculature with or without modification of its surface. Here, we present the current research milestones that so far established to reconstruct the vascular network in addition to the methods used for evaluating the efficiency of reendotheilization. Thus, this review is quite significant and will aid the researchers to know where we stand toward biofabricating a transplantable organ from decellularizd extracellular matrix.

Ultrasonicated graphene oxide enhances bone and skin wound regeneration.

In the present study, we investigated the applications of ultrasonicated graphene oxide (UGO) for bone regeneration and skin wound healing. Ultrasonication of a GO suspension increased the dispersion and stability (by increasing the zeta potential) of the GO suspension. UGO has fewer oxygen-containing groups but still displays excellent water dispersion. The UGO supension showed high biocompatibility for human fetal osteoblast (hFOB cells), human endothelial cells (EA.hy 926 cells), and mouse embryonic fibroblasts. Importantly, UGO could support cell attachment and proliferation, in addition to promoting the osteogenesis of seeded cells and the promotion of new bone formation. In addition, a 1% UGO supension enhanced cell migration in an in vitro skin scratch assay and promoted wound closure in an in vivo rat excisional skin defect model. These results showed that UGO offers a good environment for cells involved in bone and skin healing, suggesting its potential application in tissue regeneration.

Incorporation of nanoparticles into transplantable decellularized matrices: Applications and challenges.

Decellularization of tissues can significantly improve regenerative medicine and tissue engineering by producing natural, less immunogenic, three-dimensional, acellular matrices with high biological activity for transplantation. Decellularized matrices retain specific critical components of native tissues such as stem cell niche, various growth factors, and the ability to regenerate in vivo. However, recellularization and functionalization of these matrices remain limited, highlighting the need to improve the characteristics of decellularized matrices. Incorporating nanoparticles into decellularized tissues can overcome these limitations because nanoparticles possess unique properties such as multifunctionality and can modify the surface of decellularized matrices with additional growth factors, which can be loaded onto the nanoparticles. Therefore, in this minireview, we highlight the various approaches used to improve decellularized matrices with incorporation of nanoparticles and the challenges present in these applications.

Biocompatibility and hemocompatibility of efficiently decellularized whole porcine kidney for tissue engineering.

Whole kidney decellularization is a promising approach in regenerative medicine for engineering a functional organ. The reaction of the potential host depends on the biocompatibility of these decellularized constructs. Despite the proven ability of decellularized kidney scaffolds to guide cell attachment and growth, little is known about biocompatibility and hemocompatibility of these scaffolds. Our aim is to prepare decellularized kidneys of a clinically relevant size and evaluate its biocompatibility and hemocompatibility. Porcine kidneys were cannulated via the renal artery, and then perfused with 0.1% sodium dodecyl sulfate solution. Hematoxylin and eosin as well as DAPI staining confirmed cellular clearance from native kidneys in addition to preservation of the microstructure. SEM confirmed the absence of any cellular content within the scaffold, which is maintained in a well-organized 3D architecture. Decellularized kidneys retained the intact renal vasculature upon examination with contrast radiography. The essential structural extracellular matrix molecules were well-preserved. Scaffolds were susceptible to enzymatic degradation upon collagenase treatment. Scaffolds showed a good hemocompatibility when exposed to porcine blood. Decellularization was efficient to remove 97.7% of DNA from native kidneys in addition to the immunogenic and pathogenic antigens. Scaffolds did not induce the human immune response in vitro. Decellularized kidneys were non-cytotoxic to pig kidney cells (PKs). PKs were able to grow and proliferate within the decellularized renal scaffolds with maintaining a higher function than cells grown as monolayers. Thus, we have developed a rapid decellularization technique for generating biocompatible kidney scaffolds that represents a step toward development of a transplantable organ. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2034-2047, 2018.

Silver nanoparticles improve structural stability and biocompatibility of decellularized porcine liver.

No ideal cross-linking agent has been identified for decellularized livers (DLs) yet. In this study, we evaluated structural improvements and biocompatibility of porcine DLs after cross-linking with silver nanoparticles (AgNPs). Porcine liver slices were decellularized and then loaded with AgNPs (100 nm) after optimization of the highest non-toxic concentration (5 µg/mL) using Human hepatocellular carcinoma (HepG2) and EAhy926 human endothelial cell lines. The cross-linking effect of AgNPs was evaluated and compared to that of glutaraldehyde and ethyl carbodiimide hydrochloride and N-hydroxysuccinimide. The results indicated that AgNPs improved the ultra-structure of DLs' collagen fibres with good porosity and increased DLs' resistance against in vitro degradation with good cytocompatibility. AgNPs decreased the host inflammatory reaction against implanted porcine DL slices in vivo and increased the polarization of M2 macrophages. Thus, structural and functional improvements of Porcine DLs could be achieved using AgNPs.

Ultrasound-assisted green economic synthesis of hydroxyapatite nanoparticles using eggshell biowaste and study of mechanical and biological properties for orthopedic applications.

Nanostructured hydroxyapatite (HAp) is the most favorable candidate biomaterial for bone tissue engineering because of its bioactive and osteoconductive properties. Herein, we report for the first time ultrasound-assisted facile and economic approach for the synthesis of nanocrystalline hydroxyapatite (Ca10 (PO4 )6 (OH)2 ) using recycled eggshell biowaste referred as EHAp. The process involves the reaction of eggshell biowaste as a source of calcium and ammonium dihydrogen orthophosphate as a phosphate source. Ultrasound-mediated chemical synthesis of hydroxyapatite (HAp) is also carried out using similar approach wherein commercially available calcium hydroxide and ammonium dihydrogen orthophosphate were used as calcium and phosphate precursors, respectively and referred as CHAp for better comparison. The prepared materials were characterized by X-ray diffraction, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and Raman spectroscopy to determine crystal structure, particle morphology, and the presence of chemical functional groups. The nanocrystalline EHAp and CHAp were observed to have spherical morphology with uniform size distribution. Furthermore, mechanical properties such as Vickers hardness, fracture toughness, and compression tests have been studied of the EHAp and CHAp samples showing promising results. Mechanical properties show the influence of calcination at 600°C EHAp and CHAp material. After calcination, in the case of EHAp material an average hardness, mechanical strength, elastic modulus, and fracture toughness were found 552 MPa, 46.6 MPa, 2824 MPa, and 3.85 MPa m1/2 , respectively, while in the case of CHAp 618 MPa, 47.5 MPa, 2071 MPa, and 3.13 MPa m1/2 . In vitro cell studies revealed that the EHAp and CHAp nanoparticles significantly increased the attachment and proliferation of the hFOB cells. Here, we showed that EHAp and CHAp provide promising biocompatible materials that do not affect the cell viability and proliferation with enhancing the osteogenic activity of osteoblasts. Moreover, hFOB cells are found to express Osteocalcin, Osteopontin, Collagen I, Osteonectin, BMP-2 on the EHAp and CHAp bone graft. This study demonstrates the formation of pure nanocrystalline HAp with promising properties justifying the fact that the eggshell biowaste could be successfully used for the synthesis of HAp with good mechanical and osteogenic properties. These findings may have significant implications for designing of biomaterial for use in orthopedic tissue regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2935-2947, 2017.