Effects of friction stir processing and nano-hydroxyapatite on the microstructure,hardness,degradation rate and in-vitro bioactivity of WE43 alloy for biomedical applications

Nowadays,magnesium alloys are emerging in biomedical implants for their similar properties to natural bones.However,the rapid degradation of magnesium alloys in biological media hinders successful implantation.Refinement of microstructure,as well as reinforcement particles can significantly improve the degradation rate.In this work,multi-pass friction stir processing(FSP)was proposed to synthesize WE43/nano-hydroxyapatite(n HA)surface composite,the microstructure,reinforced particle distribution,micro-hardness,corrosion behavior and in-vitro bioactivity were studied.The subsequent FSP passes of WE43 alloy and WE43/n HA composite refined the grain size which was reduced by 94.29%and 95.92%(2.63 and 1.88μm,respectively)compared to base metal after three passes.This resulted in increasing the microhardness by 120%(90.86 HV0.1)and 135%(105.59 HV0.1)for the WE43 and WE43-n HA,respectively.It is found that increasing FSP passes improved the uniform distribution of n HA particles within the composite matrix which led to improved corrosion resistance and less degradation rate.The corrosion rate of the FSPed WE43/n HA composite after three passes was reduced by 38.2%(4.13 mm/year)and the degradation rate was reduced by 69.7%(2.87 mm/y).This is attributed to secondary phase(Mg24Y5and Mg41Nd5)particle fragmentation and redistribution,as well as a homogeneous distribution of n HA.Additionally,the growing Ca-P and Mg(OH)2layer formed on the surface represented a protective layer that reduced the degradation rate.The wettability test revealed a relatively hydrophilic surface with water contact angle of 49.1±2.2°compared to 71.2±2.1°for base metal.Also,biomineralization test showed that apatite layer grew after immersion 7d in simulated body fluid with atomic ratio of Ca/P 1.60 approaching the stoichiometric ratio(1.67)indicating superior bioactivity of FSPed WE43/n HA composite after three passes.These results raise that the grain refinement by FSP and introduction of n HA particles significantly improved the degradation rate and in-vitro bioactivity of WE43 alloy for biomedical applications.

Preparation and characterization of chitosan/nano-hydroxyapatite composite used as bone substitute materials

Chitosan/nano-hydroxyapatite composites with different weight ratios were prepared through a co-precipitation method using Ca（OH）2, H3PO4 and chitosan as starting materials. The properties of these composites were characterized by means of TEM, IR, XRD, TGA, burn-out tests and universal matertial testing machine. The results showed that the HA synthesized here was poorly crystalline carbonated nanometer crystals and dispersed uniformly in chitosan phase and there was no phase-separation between the two phases. The addition of n-HA resulted in a decrease of decomposing temperature of chitosan. Because of the interactions between chitosan and n-HA, the mechanical properties of these composites were improved, and the maximum value of the compressive strength was measured to be about 120MPa corresponding to the chitosan/n-HA composite with a weight ratio of 30/70.

Preparation and Characterization of Nano-hydroxyapatite/polyvinyl Alcohol Gel Composites

Nano-hydroxyapatite reinforced poly（vinyl alcohol） gel（nano-HA/PVA gel） composites has been proposed as a promising biomaterial,especially used as an articular cartilage repair biomaterial.In this paper,nano-HA/PVA gel composite was prepared by in situ synthesis method and incorporation with freeze-thaw cycle process.The microstructure and morphology were investigated by X-ray diffraction,TEM,SEM and FTIR.The results showed that the size of HA particles synthesized in PVA solution was on the nanometer scale.Both the size and crystallinity of HA particles synthesized in PVA solution decreased compared with that of HA synthesized in distilled water.The nano-HA particles were distributed in PVA matrix uniformly due to the effect of PVA solution as a dispersant while low content of HA particles in the composites.On the contrary,with high content of nano-HA particles in the composites,the particles tended to aggregate.The result of FT-IR analysis indicated that the chemical bond between nano-HA particles and PVA matrix existed.The conformation and degree of tacticity of PVA molecule changed because of the addition of HA particles.Furthermore,the interfacial strength of the composites was improved due to the interaction between nano-HA particle and PVA matrix and this was beneficial to improving the mechanical properties of the composites.

Melt-Spinning of Nano-Hydroxyapatite/Poly(ε-caprolactone)Composite Fibers for Potential Application in Bone Tissue Engineering

Nano-hydroxyapatite/poly（e-caprolactone） （nHA/PCL） composite materials are among the best candidates for application in bone tissue engineering. As the main technique to fabricate porous scaffolds, electrospilming produce scaffolds with unsatisfactory mechanical strength and limited pore size for cdi infiltration. Micron-sized fiber assembly with higher mechanical strength is qualified to structure hybrid scaffolds. In this study, nHA/PCL monofilament fibers with different mass ratios were fabricated through melt-spinning. Transmission electron microscope （TEM） was used to observe the aggregation between nHA parfides. Other characterizations including scanning electron microscopy （SEM）, attenuated total reflection Fourier transform infrared spectroscopy （ATR.FTIR） and X-ray diffraction （XRD） were done to discuss the morphology, components and crystallization of the nHA/PCL composite fibers, respectively. The influence of nHA/PCL mass ratio on the tensile properties and water contact angle of composite fibers was also studied. The SEM images show the homogeneous dispersion of nano partides in the polymer matrix. Besides, nHA content increases the tensile strength, initial modulus and hydrophillcity of the composite fibers under the premise of spinnability. This kind of fibers is strong enough to fabricate fiber assembly which may have potential application in bone tissue engineering.

PDLLA/nano-HA复合板固定下颌骨骨折愈合过程中V型胶原的表达

目的 观察采用混旋聚乳酸 /纳米羟基磷灰石 (PDLLA /nano HA)复合板 (以下简称复合板 )固定对下颌骨骨折愈合的影响。方法 对 18只新西兰兔两侧下颌骨体部截骨 ,分别采用复合板和纯PDLLA板固定。术后 2、3、4、6、12、2 4周采用免疫组织化学方法观察骨折愈合过程中V型胶原的表达。结果 术后 2～ 4周 ,复合板侧骨痂中成骨细胞、成纤维细胞及骨细胞周围基质中V型胶原高水平表达 ,呈强阳性着色 ,第 4周时达到高峰。 6周时阳性信号明显减弱 ,第 12周、2 4周成骨细胞、破骨细胞减少 ,基质呈微弱阳性着色 ,V型胶原基本无表达。PDLLA板侧 2～ 4周时与复合板侧相似 ,着色较浅 ,从第 4～ 2 4周阳性信号逐渐减弱 ,第 12周、2 4周仍可见骨痂改建征象 ,基质弱阳性着色。结论 复合板侧骨痂中V型胶原的表达强弱变化与下颌骨骨折正常愈合过程基本相符 ,复合板对骨痂的形成和改建无不利影响。

Effect of porous structure on mechanical properties of C/PLA/nano-HA composites scaffold

Nonporous and porous C/PLA/nano-HA composites were fabricated by the process of solvent blending and freeze-drying technique, and the effect of porous structure on the mechanical properties of C/PLA/nano-HA composites scaffold was investigated and analyzed. The results show that the effects of porous structure on the bending strength, modulus and curves of stress and strain were obvious. Compared with nonporous sample, the curves of stress and strain of porous sample show more rough, and alternative phenomenon of stress increase and stress relaxation appears. It is strongly suggested that the fracture model of C/PLA/nano-HA composites scaffold transforms from the local to global load due to the porous structure.

Nano-hydroxyapatite promotes cell apoptosis by co-activating endoplasmic reticulum stress and mitochondria damage to inhibit glioma growth

Despite a growing body of studies demonstrating the specific antitumor effect of nano-hydroxyapatite(n-HA),the underlying mechanism remained unclear.Endoplasmic reticulum(ER)and mitochondria are two key players in intracellular Ca^(2+)homeostasis and both require Ca^(2+)to participate.Moreover,the ER–mitochondria interplay coordinates the maintenance of cellular Ca^(2+)homeostasis to prevent any negative consequences from excess of Ca^(2+),hence there needs in-depth study of n-HA effect on them.In this study,we fabricated needle-like n-HA to investigate the anti-tumor effectiveness as well as the underlying mechanisms from cellular and molecular perspectives.Data from in vitro experiments indicated that the growth and invasion of glioma cells were obviously reduced with the aid of n-HA.It is interesting to note that the expression of ER stress biomarkers(GRP78,p-IRE1,p-PERK,PERK,and ATF6)were all upregulated after n-HA treatment,along with the activation of the pro-apoptotic transcription factor CHOP,showing that ER stress produced by n-HA triggered cell apoptosis.Moreover,the increased expression level of intracellular reactive oxygen species and the mitochondrial membrane depolarization,as well as the downstream cell apoptotic signaling activation,further demonstrated the pro-apoptotic roles of n-HA induced Ca^(2+)overload through inducing mitochondria damage.The in vivo data provided additional evidence that n-HA caused ER stress and mitochondria damage in cells and effectively restrain the growth of glioma tumors.Collectively,the work showed that n-HA co-activated intracellular ER stress and mitochondria damage are critical triggers for cancer cells apoptosis,offering fresh perspectives on ER-mitochondria targeted anti-tumor therapy.

Improving chitosan-based composite membrane by introducing a novel hybrid functional nano-hydroxyapatite with carboxymethyl cellulose and phytic acid

A functional hybrid nano-hydroxyapatite(carboxymethyl cellulose-phytic acid-n-HA,CMC-PA-n-HA)was prepared by adding CMC and PA.The results of Fourier transformation infrared spectra,X-ray diffraction,thermal gravimetric analysis and dispersion experiments indicated that the addition of CMC and PA affected the morphology,crystallinity and crystal size of hybrid n-HA,and CMC endowed hybrid n-HA with excellent dispersion.Scanning electron microscope results showed that CMC-PA-n-HA nanoparticle could be uniformly dispersed in chitosan(CS)matrix to obtain composite membrane by casting technology,so that the highest tensile strength of CMC-PA-n-HA/CS composite membrane was 69.64%and 144.45%higher than that of CS membrane and n-HA/CS composite membrane,respectively.Contact angle test showed that CMC-PA-n-HA effectively improved hydrophilicity of the CS membrane.The simulated body fluid immersion results indicated that the CMC-PA-n-HA/CS composite membrane not only exhibited good degradability but also promoted bone-like apatite deposition.The cell proliferation experiments proved that the introduction of PA made the composite membrane have better cell adhesion and proliferation ability.Antibacterial tests demonstrated that PA could effectively improve the antibacterial properties of the composite membrane,which is expected to be applied as guide bone tissue regeneration membrane.

In situ growth of nano-hydroxyapatite on multilayered Ti_(3)C_(2)T_(x) MXene as a drug carrier with superior-performance

Bone defects caused by tumor resection typically require bone repair materials to fill the defect sites.The development of multifunctional bone filling materials with integrated chemotherapy,photohermal therapy,and guided bone regeneration is very necessary and urgently needed.Herein,for the first time,the construction of novel multilayered Ti_(3)C_(2)T_(x) MXene(m-MXene)/nano-hydroxyaptite(nHAp)composites(m-MXene/nHAp)for bone repair is reported.The in situ growth of nHAp on multilayered Ti_(3)C_(2)T_(x) MXene is achieved through a facile hydrothermal method without using any organic additives.Due to the syner-gistic effects of nHAp and m-MXene,the m-MXene/nHAp composites show superior drug carrier perfor-mance with ultra-high drug loading capacity and ultra-long drug sustained release time.The molecular dynamics simulation results indicate that both Ti_(3)C_(2)T_(x) MXene and HAp show many adsorption sites and high binding energy with DOX.Moreover,the m-MXene/nHAp composites possess high photothermal conversion efficiency and excellent photothermal stability.The in situ growth of nano-HAp can signifi-cantly improve the biocompatibility of the m-MXene.The as-prepared multifunctional m-MXene/nHAp composites in this work can be used as bone filling powder and have great potential in bone defect reconstruction caused by bone tumor.

混旋聚乳酸/纳米羟基磷灰石板在颧上颌骨骨折内固定中的应用

目的 :探讨混旋聚乳酸 /纳米羟基磷灰石 (PDLLA/nano -HA)复合板 (简称复合板 )在颧上颌骨骨折内固定中的应用。方法 :2例颧上颌骨骨折患者均用复合板、钛钉固定 ,术后 1～ 3周观察骨折愈合情况及局部组织反应。结果 :2例患者创口均Ⅰ期愈合 ,局部组织无明显排斥反应和炎性反应。骨折固位稳定 ,无异常骨活动。咬合关系恢复良好 ,张口度无异常 ,面部外形恢复正常 ,均获得满意效果。结论 :复合板既具有金属坚强内固定系统的优点 ,又克服了金属内固定系统的应力遮挡作用 ,适用于颧上颌骨骨折。

Nano-hydroxyapatite accelerates vascular calcification via lysosome impairment and autophagy dysfunction in smooth muscle cells

Vascular calcification(VC)is a common characteristic of aging,diabetes,chronic renal failure,and atherosclerosis.The basic component of VC is hydroxyapatite(HAp).Nano-sized HAp(nHAp)has been identified to play an essential role in the development of pathological calcification of vasculature.However,whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear.We discovered that nHAp existed both in vascular smooth muscle cells(VSMCs)and their extracellular matrix(ECM)in the calcified arteries from patients.Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery.nHAp stimulated osteogenic differentiation and accelerated mineralization of VSMCs in vitro.Synthetic nHAp could also directly induce VC in vivo.Mechanistically,nHAp was internalized into lysosome,which impaired lysosome vacuolar H+-ATPase for its acidification,therefore blocked autophagic flux in VSMCs.Lysosomal re-acidification by cyclic-3′,5′-adenosine monophosphate(cAMP)significantly enhanced autophagic degradation and attenuated nHAp-induced calcification.The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit.Inhibition of exosome release in VSMCs decreased calcium deposition.Altogether,our results demonstrated a repressive effect of nHAp on lysosomal acidification,which inhibited autophagic degradation and promoted a conversion of the accumulated autophagic vacuoles into exosomes that were loaded with undissolved nHAp,Ca^(2+),Pi and ALP.These exosomes bud off the plasma membrane,deposit within ECM,and form calcium nodules.Vascular calcification was thus accelerated by nHAP through blockage of autophagic flux in VSMCs.

Biocompatibility of nano-hydroxyapatite/Mg-Zn-Ca alloy composite scaffolds to human umbilical cord mesenchymal stem cells from Wharton's jelly in vitro

Seeding cells and scaffolds play pivotal roles in bone tissue engineering and regenerative medicine.Wharton’s jelly-derived mesenchymal stem cells(WJCs)from human umbilical cord represent attractive and promising seeding cells in tissue regeneration and engineering for treatment applications.This study was carried out to explore the biocompatibility of scaffolds to seeding cells in vitro.Rod-like nano-hydroxyapatite(RN-HA)and flake-like micro-hydroxyapatite(FM-HA)coatings were prepared on Mg-Zn-Ca alloy substrates using micro-arc oxidation and electrochemical deposition.WJCs were utilized to investigate the cellular biocompatibility of Mg-Zn-Ca alloys after different surface modifications by observing the cell adhesion,morphology,proliferation,and osteoblastic differentiation.The in vitro results indicated that the RN-HA coating group was more suitable for cell proliferation and cell osteoblastic differentiation than the FM-HA group,demonstrating better biocompatibility.Our results suggested that the RN-HA coating on Mg-Zn-Ca alloy substrates might be of great potential in bone tissue engineering.

Comparative evaluation of the physicochemical properties of nano-hydroxyapatite/collagen and natural bone ceramic/collagen scaffolds and their osteogenesis-promoting effect on MC3T3-E1 cells

The use of various types of calcium phosphate has been reported in the preparation of repairing materials for bone defects.However,the physicochemical and biological properties among them might be vastly different.In this study,we prepared two types of calcium phosphates,nano-hydroxyapatite(nHA)and natural bone ceramic(NBC),into 3D scaffolds by mixing with type I collagen(CoL),resulting in the nHA/CoL and NBC/CoL scaffolds.We then evaluated and compared the physicochemical and biological properties of these two calcium phosphates and their composite scaffold with CoL.Scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS),Fourier-transform infrared spectroscopy(FTIR),X-ray diffraction spectroscopy(XRD)and compressive tests were used to,respectively,characterize the morphology,composition,distribution and the effect of nHA and NBC to collagen.Next,we examined the biological properties of the scaffolds using cytotoxicity testing,flow cytometry,immunofluorescence staining,biocompatibility testing,CCK-8 assays and RT-PCR.The results reflected that the Ca2t released from nHA and NBC could bind chemically with collagen and affect its physicochemical properties,including the infrared absorption spectrum and compression modulus,among others.Furthermore,the two kinds of scaffolds could promote the expression of osteo-relative genes,but showed different gene induction properties.In short,NBC/CoL could promote the expression of early osteogenic genes,while nHA/CoL could upregulate late osteogenic genes.Conclusively,these two composite scaffolds could provide MC3T3-E1 cells with a biomimetic surface for adhesion,proliferation and the formation of mineralized extracellular matrices.Moreover,nHA/CoL and NBC/CoL had different effects on the period and extent ofMC3T3-E1 cell mineralization.

Magnesium/Nano-hydroxyapatite Composite for Bone Reconstruction: The Effect of Processing Method

Nano-ceramic particles can serve as reinforcing agents for metallic materials to improve their mechanical properties.However,it is important to ensure chemical compatibility between the matrix and particles.In the present study,magnesium composites with and without nano-hydroxyapatite(nHA)particles were fabricated for bone reconstruction applications.Two different techniques were used,Conventional Sintering(CS)of powder compacts and Spark Plasma Sintering(SPS)of pre-compacted powder.Results showed that a 10 wt%addition of nHA particles to magnesium,followed by SPS improved the compression strength by 27%.CS did not lead to any significant improvement compared to SPS processing.X-ray diffraction data after CS revealed the formation of unfavorable phases due to chemical reactions between nHA particles and the magnesium matrix,while these phases were absent after SPS processing.The mechanical properties of the specimens fabricated by CS were much inferior to those processed using SPS.The shorter processing time associated with SPS leaded to reduced interaction between nHA particles and the Mg-matrix,compared to CS.

Preparation and evaluation of nano-hydroxyapatite/poly(styrene-divinylbenzene) porous microsphere for aspirin carrier

A novel vehicle for the delivery of aspirin （ASA） was prepared from porous nano-hydroxyapafite/poly（styrene-divinylbenzene） [nano-HAP/P（St-DVB）] composite microspheres by grafting nano-HAP [Ca10（PO4）6（OH）2] onto porous P（St-DVB） micro- spheres. Four types of porous composite microspheres were prepared, each with different nano-HAP contents. The ASA-loaded composite microspheres prepared with 10% and 15% nano-HAP （mass ratio） exhibited excellent buoyancy with relatively short instantaneous floating time （within l0 min） and a long sustained floating time （12 h） in simulated gastric juice. They also offered good sustained release of ASA （up to 8 h）. Furthermore, these composite microspheres displayed good buff- ering capacity that prevented the buildup of acidity caused by hydrolysis of ASA, keeping the pH of gastric juice within the normal range （pH 0.9 to 1.5）. The results showed that porous nano-HAP/P（St-DVB） composite microspheres prepared with 10% and 15% nano-HAP could be used as a novel drug carrier for ASA, providing a sustained release dose without leading to stomach irritation, a side effect that is often associated with ASA medication.

Effect of Antibacterial Enoxacin on the Properties of Injectable Nano-hydroxyapatite/Polyurethane Cement for Bone Repairing

Biomaterial-associated infection(BAI)is a kind of serious post-operative complication in orthopaedic surgery.Antibiotic-loaded bone cement shines a light on BAI prevention for convenient manipulation and complex filling.To this aim,we designed an antibacterial bone cement based on Nano-hydroxyapatite/Polyurethane(PUHA)loading with antibiotic Enoxacin(EN).The distinct shear-thinning behavior of the prepolymers was observed,indicating a good injectability.The PUHA bone cement possessed a suitable curing speed,and the addition of EN might slightly expedite the curing process and enhance the mechanical properties.The EN release profile indicated that the EN-loaded bone cement could reach the minimum inhibitory concentration in 2 h,and sustainedly released EN for almost 8 days,exhibiting an antibacterial delivery potential.Antibacterial test further confirmed the antibacterial ability of EN-loaded bone cement is in a dose-dependent manner.However,the osteogenic performance of drug-loaded bone cement with high dosage is not as good as antibacterial activity.When the EN concentration of antibacterial cement was lower than 32μg·mL^(-1),the proliferation and osteogenic differentiation of rat mesenchymal stem cells could be significantly promoted.Overall,this study verified the potential of the EN-loaded PUHA bone cement in anti-infection and osteogenesis for bone repairing.

Nano-hydroxyapatite(n-HA)involved in the regeneration of rat nerve injury triggered by overloading stretch

Damage of axon and glial scars formation both inhibit nerve regenerative growth during nerve injury.In addition,mechanical stretch at high displacement rates of 10%tensile strain can cause marked nerve injury,it is important for finding a proper nano biomaterial to repair nerve injury.Nano-hydroxyapatite(n-HA)has excellent biocompatibility and high bioactivity,which is a good candidate for biomedical engineering applications.But the certain mechanism of n-HA on the injured nerve is seldom reported.In this study,we determined the role of n-HA on the mechanical stretch-induced nerve injury at adult rat spine.Mechanical stretch under strain 10%at displacement rates of 60 mm/min can cause marked broken vessels and edema in spinal cord and dorsal root ganglion tissue in haematoxylin-eosin(HE)staining.However,n-HA application can reverse hemorrhage and edema triggered by high rates of 60 mm/min stretch.Moreover,n-HA can promote positive staining of Netrin-1 increase significantly in spinal cord and dorsal root ganglion tested by immunohistochemistry(IHC)staining.In general,our study indicated that n-HA can repair mechanical stretch-induced nerve injury,it may provide a new approach to block injury and accelerate nerve regeneration in future.

Blood Compatibility of Nano-Hydroxyapatite Dispersed Using Various Agents

Nano-hydroxyapatite (nHAP),dispersed with three kinds of dispersants (heparin sodium,polyacrylic sodium and wa-ter),reacted with red blood cell,Bel-7402 tumor cell,to compare their dispersing efficiency against nHAP from one another. The blood compatibility of nHAP is also determined by blood solubil-ity experiment so that the capability of different dispersant dis-persing against nHAP of different concentration and the relation between nHAP and blood compatibility have been determined. The inhibiting function of Bel-7402 against tumor cells is deter-mined with the MTT staining method. The study result shows that heparin sodium has the best dispersing efficiency for nHAP with-out the phenomenon of hemolysis.

Advancements of Nano-Hydroxyapatite in Sports Medicine from Bone Injury Perspectives

Nanotechnology has revolutionized the field of biology and medicine in the 21 st century.Bone injury incidences during sports activities are common,and they are traditionally treated with allogeneic grafting,a common clinical practice but limited by the quality of the graft and some side effects.Nano-hydroxyapatite(nHA)is considered as an ideal bone graft material owing to its bone-like structure,excellent biological activity,bone conductivity,non-toxicity,and non-immunogenicity.nHA and its composite materials have been found suitable for the adhesion,proliferation,and differentiation of mesenchymal stem cells,which leads to their potential applications in treating the bone injury.In this review,we classified different applications of nHA to explore the role of these materials in bone repair and tendon healing,highlighting the superior characteristics of nanomaterials in the treatment of bone injury,hoping to provide ideas for nHA applied to clinical practices for the treatment of bone injury.

Extraction of Natural Nanostructured Hydroxyapatite from Pacific Cod(Gadus macrocephalus)Bone with a Thermostable Collagenolytic Protease and Its ex vivo Intestinal Bioavailability

Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.