High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells

BACKGROUND Cartilage defects are some of the most common causes of arthritis.Cartilage lesions caused by inflammation,trauma or degenerative disease normally result in osteochondral defects.Previous studies have shown that decellularized extracellular matrix(ECM)derived from autologous,allogenic,or xenogeneic mesenchymal stromal cells(MSCs)can effectively restore osteochondral integrity.AIM To determine whether the decellularized ECM of antler reserve mesenchymal cells(RMCs),a xenogeneic material from antler stem cells,is superior to the currently available treatments for osteochondral defects.METHODS We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70%confluence;50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition.Decellularized sheets of adipocyte-derived MSCs(aMSCs)and antlerogenic periosteal cells(another type of antler stem cells)were used as the controls.Three weeks after ascorbic acid stimulation,the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints.RESULTS The defects were successfully repaired by applying the ECM-sheets.The highest quality of repair was achieved in the RMC-ECM group both in vitro(including cell attachment and proliferation),and in vivo(including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues).Notably,the antler-stem-cell-derived ECM(xenogeneic)performed better than the aMSC-ECM(allogenic),while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells.CONCLUSION Decellularized xenogeneic ECM derived from the antler stem cell,particularly the active form(RMC-ECM),can achieve high quality repair/reconstruction of osteochondral defects,suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

Deer antler stem cell niche: An interesting perspective

In recent years,there has been considerable exploration into methods aimed at enhancing the regenerative capacity of transplanted and/or tissue-resident cells.Biomaterials,in particular,have garnered significant interest for their potential to serve as natural scaffolds for cells.In this editorial,we provide commentary on the study by Wang et al,in a recently published issue of World J Stem Cells,which investigates the use of a decellularized xenogeneic extracellular matrix(ECM)derived from antler stem cells for repairing osteochondral defects in rat knee joints.Our focus lies specifically on the crucial role of biological scaffolds as a strategy for augmenting stem cell potential and regenerative capabilities,thanks to the establishment of a favorable microenvironment(niche).Stem cell differen-tiation heavily depends on exposure to intrinsic properties of the ECM,including its chemical and protein composition,as well as the mechanical forces it can generate.Collectively,these physicochemical cues contribute to a bio-instructive signaling environment that offers tissue-specific guidance for achieving effective repair and regeneration.The interest in mechanobiology,often conceptualized as a form of“structural memory”,is steadily gaining more validation and momen-tum,especially in light of findings such as these.

Injectable hydrogel made from antler mesenchyme matrix for regenerative wound healing via creating a fetal-like niche

BACKGROUND Scar formation and loss of cutaneous appendages are the greatest challenges in cutaneous wound healing.Previous studies have indicated that antler reserve mesenchyme(RM)cells and their conditioned medium improved regenerative wound healing with partial recovery of cutaneous appendages.AIM To develop hydrogels from the antler RM matrix(HARM)and evaluate the effect on wound healing.METHODS We prepared the hydrogels from the HARM via enzymatic solubilization with pepsin.Then we investigated the therapeutic effects of HARM on a full-thickness cutaneous wound healing rat model using both local injections surrounding the wound and topical wound application.RESULTS The results showed that HARM accelerated wound healing rate and reduced scar formation.Also,HARM stimulated the regeneration of cutaneous appendages and blood vessels,and reduced collagen fiber aggregation.Further study showed that these functions might be achieved via creating a fetal-like niche at the wound site.The levels of fetal wound healing-related genes,including Collagen III and TGFβ3 treated with HARM were all increased,while the expression levels of Collagen I,TGFβ1,and Engrailed 1 were decreased in the healing.Moreover,the number of stem cells was increased in the fetal-like niche created by HARM,which may contribute to the regeneration of cutaneous appendages.CONCLUSION Overall,we successfully developed an injectable hydrogel made from antler RM matrix for the regenerative repair of full-thickness cutaneous wounds.We uncovered the molecular mechanism of the hydrogels in promoting regenerative wound healing,and thus pave the way for HARM to be developed for the clinic use.

Discussion on the Main Reasons for a Large Number of Père David s Deer ( Elaphurus davidianus ) with Abnormal Antler Shedding

[Objectives]This study was conducted to find out the causes of abnormal antler shedding in Père David s deer(Elaphurus davidianus).[Methods]Abnormally-shed antlers were compared with normally-shed antlers in terms of light condition,antler development and bone nutritional status during the abnormal shedding season in the growth area of Père David s deer.[Results]Abnormally-shed antlers had no significant differences in the development of shed antlers,or even in the overall composition of antlers,from those of normal Père David s deer.[Conclusions]Insufficient light was the main cause of abnormal antler shedding in Père David s deer.

Regulations of Antler Generation and Regeneration by Nutritional Factors

Antler, one of the secondary sex characteristics of male deer, is a highly valued traditional Chinese medicine. The generation and regeneration of deer antler is regulated not only by hormones, but also by nutritional factors. Nutritional factors have great influences on pedicle generation, first antler generation and antler development. Therefore, this paper summarizes recent advances in the study of the influences of nutritional factors on antler generation and regeneration, analyzes the pathways regulating antler generation and regeneration by nutritional factors, and proposes thai nutritional factors regulale antler regeneralion by direct pathway and indirect pathway.

Antler stem cells and their potential in wound healing and bone regeneration

Compared to other vertebrates,the regenerative capacity of appendages in mammals is very limited.Deer antlers are an exception and can fully regenerate annually in postnatal mammals.This process is initiated by the antler stem cells(AnSCs).AnSCs can be divided into three types:(1)Antlerogenic periosteum cells(for initial pedicle and first antler formation);(2)Pedicle periosteum cells(for annual antler regeneration);and(3)Reserve mesenchyme cells(RMCs)(for rapid antler growth).Previous studies have demonstrated that AnSCs express both classic mesenchymal stem cells(MSCs)and embryonic stem cells(ESCs),and are able to differentiate into multiple cell types in vitro.Thus,AnSCs were defined as MSCs,but with partial ESC attributes.Near-perfect generative wound healing can naturally occur in deer,and wound healing can be achieved by the direct injection of AnSCs or topical application of conditioned medium of AnSCs in rats.In addition,in rabbits,the use of both implants with AnSCs and cell-free preparations derived from AnSCs can stimulate osteogenesis and repair defects of bone.A more comprehensive understanding of AnSCs will lay the foundation for developing an effective clinical therapy for wound healing and bone repair.

Combination of Supercritical Fluid Extraction with Ultrasonic Extraction for Obtaining Sex Hormones and IGF-1 from Antler Velvet

Supercritical CO2 (SC-CO2) extraction technology and ultrasonic technology were used to extract two active sex hormones, estradiol and progesterone, and insulin-like growth factor-1 (IGF-1) from antler velvet. The effects of SC-CO2 extraction condition on the extraction yield and content of sex hormones, the ultrasonic extrac-tion condition on the content of IGF-1 and the SC-CO2 extraction condition on the activity remaining of IGF-1 were studied. The optimal conditions were obtained. The experimental results showed that, in presence of 75% ethanol as the co-solvent, the mean yield and content of estradiol and progesterone were 87.67 pg·g-1 and 1224.10 pg·g-1, 12.38 ng·g-1 and 354.06 ng·g-1, respectively, with extraction pressure of 30 MPa, temperature of 35°C, extraction time of 30 min and CO2 consumption of 15 L·g-1 at the flow rate of 2.0 L·min-1. The highest content of IGF-1 was 7425.75 ng·g-1 antler velvet residue, when the pH10 ammonia-ammonium chloride buffer solution was used as the solvent, the ratio of solvent to sample was 20/1 (volume/mass), the extraction temperature was 0-35°C, and the ex-traction time was 4×15 min. Under these conditions, 93.68% activity remaining of IGF-1 in the residue was ob-tained, while little IGF-1 activity exists in traditional residue. The experimental results indicate that the technology of SC-CO2 with co-solvent is of advantage for getting high content sexual hormones and keeping high activity of IGF-1 in the residue, which can not be achieved by traditional extraction methods.

Determination of Sex Hormones in Antler Velvet by High Performance Liquid Chromatography Tandem Mass Spectrometry

Eighteen sex hormones in antler velvet were determined by high performance liquid chromatography tandem mass spectrometry.The solid phase extraction was applied to eliminating the matrix effect.The experimental conditions were examined and optimized.Under the optimal conditions,the proposed method provides the good linearities and determination limits（0.2―1.0 μg/kg） of the analytes investigated.The recoveries ranging from 72.3% to 149.5% were obtained for the target analytes at two concentration levels.This method was applied to the determination of eighteen sex hormones in different kinds of antler velvet samples and the obtained results are satisfactory.The results indicate that the proposed method is suitable for the determination of sex hormones in antler velvet samples.

Antlers Are Shaped as Elements of Concave Surfaces that Reflect and Concentrate Incoming Sounds

In 1917 D’Arcy Thompson observed that the pair of antlers of a red deer, a sambur or a wapiti Make up between them a single surface that is more or less spherical or occasionally an ellipsoidal surface. The palmated antlers of the moose are explored to provide an exam-ple of how antlers can be instrumental in lowering the threshold sensitivity of hearing.

Therapeutic effects of velvet antler polypeptides on hepatic fibrosis in rats

OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides(VVAPs)in CCl4-induced experimental hepatic fibrosis in rats.METHODS Anti-hepatic fibrosis properties of VAPs were tested by Subcutaneous injection(SC)into male Wistar rats of CCl4- induced experimental hepatic fibrosis.After SC injections for 45 consecutive days at doses of 5mg·kg-1(low dose,VAPsL),10mg·kg-1(mid-dose,VAPsM)and 20mg·kg-1(high-dose,VAPsH),the rats were sacrificed and the various indicators were evaluated and tested.Observed hepatic cells degeneration and necrosis,inflammatory infiltration and levels of serum enzymes to assess treatment of VAPs;The expression levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),MDA,and hydroxyproline(HYP)in liver tissue were analyzed;RT-PCR analysis was carried out to detect the expression levels of matrix metalloproteinases2(MMP-2)and tissue inhibitor of metalloproteinases 1(TIMP-1)in liver tissue.RESULTS VAPs has obvious anti-hepatic fibrosis effects.Hepatocyte swelling,fatty degeneration was significantly reduced,reducing infiltration of inflammatory cells.Release of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)decreased significantly,reduction of hyaluronic acid(HA)and laminin(LN)obviously,at the same time,the content of total protein and albumin increased significantly in serum.Activity of SOD and GSH-Px was significantly raised and the content of MDA and HYP was reduced significantly in liver tissue.Expression levels of MMP-2and TIMP-1 mRNA in liver were decreased significantly.These improvements were more significant in high-dos and mid-dose groups(P<0.05 or P<0.01 vs model group).CONCLUSION These findings suggest VAPs can significant treat the hepatic fibrosis,which may be due to protect liver cells and improve liver functions by hydroxyl radical scavenging activity and great effect of antioxidation,and decrease the gene expression of MMP-2,improving exist-environment of liver cells and decreasing the gene expression of TIMP-1,prompting degradation of extracellular matrix.

Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

ANXA2（AnnexinA2）, a calcium-dependent phospholipid bind- ing protein, is involved in various Ca2＋-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer （Cervus nippon hortulomm）; the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcdptase polymerase chain reaction （RT-PCR） techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the opan-reading frame （OR.F） encoding 339 amino acids; its relative mo- lecular weight was 38.3 kDa; and isoelectrie point was 6.72. Sequence analysis indicates that the protein includes four conserved tan- dem-duplication ANX domains. The gene-aceession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re- veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza- tion.

TLC Identification of Schisandra Chinensis Liquid Extract and Antler Essence in Qianglinaoqingsu Tablets

[Objectives]To study the quality standard of Chinese drug in compound Qianglinaogingsu Tablets(Fructus Schisandrae Chinensis and Coru Cervi Pantotrichum).[Methods]The thin layer chromatography(TLC)differentiation method was established for Fructus Schisandrae Chinensis and Cornu Cervi Pantotrichum.[Results]TLC spots developed were fairly clear,and the blank test showed no interference.[Conclusions]The method is fast,simple and can be used for quality control of Qianglinaoqingsu Tablets.

Kanglu Brand Pilose Antler Nourishing Cream

Kanglu Brand Pilose Antler Nourishing Cream, developed by the Navy Anti-aging Research Institute and the Hainan Fengmu Deer Breeding Farm, is a kind of high-tech nourishing cream. The cream is produced from the fresh pilose antler of younger deer, and contains the quintessence of pilose antlers——the total fat and SOD. The SOD is regarded as the best anti-aging agent

Deer antler renewal gives insights into mammalian epimorphic regeneration

Deer antlers are the only known mammalian organ that,once lost,can fully grow back naturally.Hence,the antler offers a unique opportunity to learn how nature has solved the problem of mammalian epimorphic regeneration(EpR).Comprehensive comparisons amongst different types of EpR reveal that antler renewal is fundamentally different from that in lower vertebrates such as regeneration of the newt limb.Surprisingly,antler renewal is comparable to wound healing over a stump of regeneration-incompetent digit/limb,bone fracture repair,and to a lesser extent to digit tip regeneration in mammals.Common to all these mammalian cases of reaction to the amputation/mechanical trauma is the response of the periosteal cells at the distal end/injury site with formation of a circumferential cartilaginous callus(CCC).Interestingly,whether the CCC can proceed to the next stage to transform to a blastema fully depends on the presence of an interactive partner.The actual form of the partner can vary in different cases with the nail organ in digit tip EpR,the opposing callus in bone fracture repair,and the closely associated enveloping skin in antler regeneration.Due to absence of such an interactive partner,the CCC of a mouse/rat digit/limb stump becomes involuted gradually.Based on these discoveries,we created an interactive partner for the rat digit/limb stump through surgically removal of the interposing layers of loose connective tissue and muscle between the resultant CCC and the enveloping skin after amputation and by forcefully bonding two tissue types tightly together.In so doing partial regeneration of the limb stump occurred.In summary,if EpR in humans is to be realized,then I envisage that it would be more likely in a manner akin to antler regeneration rather to that of lower vertebrates such as newt limbs.

Comparison of Structure and Biological Activity of Natural Polypeptide from Velvet Antlers of Cervus elaphus with Those of Synthesized Polypeptide

Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and re- versed-phase high-performance liquid chromatography（RP-HPLC） were used to isolate and purify the natural poly- peptide from velvet antler（nVAP） of Cervus elaphus（C, elaphus）, which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4--50 gg/mL, nVAP promotes mitosis in epidermal ceils, chondrocytes and NIH3T3 fibroblasts pri- marily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide（sVAP） according to its primary struc- ture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectro- metry（MALDI-TOF-MS） was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAE Bioactivity test shows that at a dose of 5--40 μg/mL, sVAP promotes the pro- liferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon（C, nippon） and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus, sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler polypeptide of C. elaphus.

鹿茸菇多糖的提取工艺优化及其对大鼠运动性骨骼肌损伤影响的研究

采用响应面法优化提取鹿茸菇中的多糖,考察酶添加量、酶解温度、酶解时间对鹿茸菇多糖得率的影响,并研究其对大鼠运动性骨骼肌损伤的影响。结果表明:添加量2.1%、酶解温度50.4℃、酶解时间98 min,据此调整后的工艺参数重复3次实验,得出鹿茸菇多糖平均得率为9.24%,与预测值接近。大鼠运动试验表明,灌胃鹿茸菇多糖的大鼠超氧化物歧化酶、谷胱甘肽过氧化物酶水平明显提高,丙二醛含量降低,乳酸脱氢酶、肌酸激酶水平明显降低,表明鹿茸菇多糖具有良好的抗氧化能力,可以降低骨骼肌细胞膜的通透性,从而降低大鼠力竭运动之后产生的自由基对其骨骼肌造成的损伤,进而提高了大鼠的抗氧化能力。

鹿茸多肽对顺铂所致卵巢早衰大鼠卵巢功能的保护作用及其机制

目的:探讨鹿茸多肽(VAP)对顺铂(DDP)致卵巢早衰模型大鼠的影响及机制。方法:取动情周期正常的雌性大鼠并将其随机分为对照组、模型组、VAP干预组,每组各10只。模型组和VAP干预组通过连续20 d腹腔注射DDP(4 mg·kg^(−1)·d^(−1))建立卵巢早衰大鼠模型,干预组同时连续20 d灌胃VAP(400 mg·kg^(−1)·d^(−1))。HE染色观察卵巢形态学表现,并对各级发育卵泡计数。ELISA检测血清雌二醇(E2)、促卵泡生成素(FSH)、抗穆勒管激素(AMH)水平。生化法检测卵巢组织中谷胱甘肽(GSH)和丙二醛(MDA)水平。探针标记法检测卵巢组织中活性氧(ROS)水平。普鲁士蓝染色观察卵巢组织中铁蓄积情况并检测亚铁离子(Fe^(2+))水平。免疫组化和Western blot检验卵巢组织中溶质载体家族7成员11(SCL7A11)、谷胱甘肽过氧化酶4(GPX4)和酰基辅酶A合成酶长链家族成员4(ACSL4)的表达。通过生育力监测观察各组大鼠生育能力。结果:与对照组相比,模型组卵巢萎缩,铁蓄积(P<0.01),各级发育卵泡和总卵泡减少(P<0.05),闭锁卵泡增多(P<0.01),产仔数减少(P<0.01),血清E2、AMH水平降低(P<0.01),FSH水平提高(P<0.01),卵巢组织中GSH水平、SCL7A11和GPX4蛋白表达均降低(P<0.05),Fe^(2+)、MDA和ROS水平及ACSL4蛋白表达均升高(P<0.01)。与模型组相比,VAP能改善DDP导致的上述指标改变(P<0.01)。结论:鹿茸多肽可改善顺铂致卵巢早衰大鼠的卵巢功能、维持正常的卵泡发育并提高其生育能力,机制可能与脂质过氧化反应和铁死亡有关。

多模态影像学方法评估布依药鹿角壮骨煎液的骨折疗效

目的 运用多模态影像学方法评估布依药鹿角壮骨煎液的骨折疗效。方法通过手术方式制作新西兰白兔左下肢胫骨中下段骨折模型30只,随机分成实验组15只给予布依药鹿角壮骨煎液灌胃治疗,对照组15只给予等量0.9%生理盐水灌胃治疗。在造模后当天、第7d、15d、21d、30d、42d随机选择2只模型兔分别行常规DR摄片、常规CT及双能量CT扫描,并对CT扫描数据行冠矢状位及骨髓成像后处理,对比不同影像学方法对实验组及对照组骨折端的对显示情况。结果与对照组比较,实验组在X平片、CT平扫、双能量骨髓成像评估中均有统计学差异(P>0.05),且CT平扫结合双能量骨髓成像对骨折愈合评估更加敏感、准确;对于骨折治愈周期比较,实验组较对照组愈合周期明显缩短,7天时间点骨折的修复评分较对照组提高97.3%。结论布依药鹿角壮骨煎液可促进骨痂生长,缩短骨折病程,且CT平扫结合双能骨髓成像对骨折愈合的评估更加敏感、准确。

鹿茸肉苁蓉复方缓解运动小鼠疲劳的功效研究

分析鹿茸肉苁蓉复方缓解运动小鼠疲劳的功效与机理,为提高人的运动耐力及缓解疲劳的深入研究提供理论基础。本试验分为四组,每组各10只小鼠。连续30 d对小鼠经口灌胃后测定小鼠的负重游泳时间、血乳酸、血清尿素氮、肝糖原以及红细胞携氧能力。结果表明,三个给药组小鼠的负重游泳时间均有所延长,说明单独使用马鹿茸、肉苁蓉、黄芪、枸杞能延长小鼠的负重游泳时间,具有缓解疲劳的作用。在试验一组配方的基础上增加当归、玫瑰花、丁香可显著缓解小鼠的疲劳,可能与减少血乳酸、血清尿素氮的堆积以及提高红细胞携氧能力有关。

鹿茸标准鉴别技术进展研究

鹿茸是一种具有重要药用价值的中药材,具有多种功效和广泛的临床应用。然而,由于市场上出现了大量的假冒伪劣产品,对鹿茸的鉴别技术的研究变得十分重要。本文综述了近年来鹿茸的鉴别技术进展,包括传统的鉴别方法以及现代分子生物学和化学分析技术等。同时,还对当前鹿茸鉴别技术的挑战和未来发展方向进行了讨论。