Mesoporous Bioglass/Silk Fibroin Scaffolds as a Drug Delivery System: Fabrication, Drug Loading and Release in vitro and Repair Calvarial Defects in vivo

The potential of combining bioactive glass（MBG） and silk fibroin（SF） together as a new drug delivery system was evaluated. The three-dimensional porous scaffolds were selected as the form of SF, and sol-gel method was adopted to fabricate MBG in this study. The characteristic of the synthesized material was measured by transmission electron microscopy and scanning electron microscopy. In vitro evaluation of drug delivery was carried out in terms of drug loading and drug release. And aspirin was chosen as the drug for scaffolds to carry out in vitro tests and repair BALB/C mice calvarial defects. Bone formation was examined by microcomputed tomography. The experimental results show that MBG/silk scaffolds have better physiochemical properties compared with silk scaffolds. In comparison to pure silk scaffolds, MBG/silk scaffolds enhance the drug loading efficiency, release rate in vitro and promote bone regeneration in vivo. Thus we conclude that MBG/silk scaffold is a more efficient drug delivery system than pure silk scaffolds.

Early therapeutic effect of platelet-rich fibrin combined with allogeneic bone marrow-derived stem cells on rats’ critical-sized mandibular defects

BACKGROUND Critically sized bone defects represent a significant challenge to orthopaedic surgeons worldwide.These defects generally result from severe trauma or resection of a whole large tumour.Autologous bone grafts are the current gold standard for the reconstruction of such defects.However,due to increased patient morbidity and the need for a second operative site,other lines of treatment should be introduced.To find alternative unconventional therapies to manage such defects,bone tissue engineering using a combination of suitable bioactive factors,cells,and biocompatible scaffolds offers a promising new approach for bone regeneration.AIM To evaluate the healing capacity of platelet-rich fibrin(PRF)membranes seeded with allogeneic mesenchymal bone marrow-derived stem cells(BMSCs)on critically sized mandibular defects in a rat model.METHODS Sixty-three Sprague Dawley rats were subjected to bilateral bone defects of critical size in the mandibles created by a 5-mm diameter trephine bur.Rats were allocated to three equal groups of 21 rats each.Group I bone defects were irrigated with normal saline and designed as negative controls.Defects of group II were grafted with PRF membranes and served as positive controls,while defects of group III were grafted with PRF membranes seeded with allogeneic BMSCs.Seven rats from each group were killed at 1,2 and 4 wk.The mandibles were dissected and prepared for routine haematoxylin and eosin(HE)staining,Masson's trichrome staining and CD68 immunohistochemical staining.RESULTS Four weeks postoperatively,the percentage area of newly formed bone was significantly higher in group III(0.88±0.02)than in groups I(0.02±0.00)and II(0.60±0.02).The amount of granulation tissue formation was lower in group III(0.12±0.02)than in groups I(0.20±0.02)and II(0.40±0.02).The number of inflammatory cells was lower in group III(0.29±0.03)than in groups I(4.82±0.08)and II(3.09±0.07).CONCLUSION Bone regenerative quality of critically sized mandibular bone defects in rats was better promoted by PRF membranes seeded with BMSCs than with PRF membranes alone.

Selective serotonin re-uptake inhibitor sertraline inhibits bone healing in a calvarial defect model

Bone wound healing is a highly dynamic and precisely controlled process through which damaged bone undergoes repair and complete regeneration. External factors can alter this process, leading to delayed or failed bone wound healing. The findings of recent studies suggest that the use of selective serotonin reuptake inhibitors(SSRIs) can reduce bone mass, precipitate osteoporotic fractures and increase the rate of dental implant failure. With 10% of Americans prescribed antidepressants, the potential of SSRIs to impair bone healing may adversely affect millions of patients’ ability to heal after sustaining trauma. Here, we investigate the effect of the SSRI sertraline on bone healing through pre-treatment with(10 mg·kg-1sertraline in drinking water, n = 26) or without(control, n = 30) SSRI followed by the creation of a 5-mm calvarial defect. Animals were randomized into three surgical groups:(a) empty/sham,(b) implanted with a DermaMatrix scaffold soak-loaded with sterile PBS or(c) DermaMatrix soak-loaded with542.5 ng BMP2. SSRI exposure continued until sacrifice in the exposed groups at 4 weeks after surgery. Sertraline exposure resulted in decreased bone healing with significant decreases in trabecular thickness, trabecular number and osteoclast dysfunction while significantly increasing mature collagen fiber formation. These findings indicate that sertraline exposure can impair bone wound healing through disruption of bone repair and regeneration while promoting or defaulting to scar formation within the defect site.

Evaluation of Bone Regeneration of Simvastatin Loaded Chitosan Nanofiber Membranes in Rodent Calvarial Defects

Chitosan nanofiber membranes have been known to have a high degree of biocompatibility and support new bone formation with controllable biodegradation. The surface area of these membranes may allow them to serve as local delivery carriers for different biologic mediators. Simvastatin, a drug commonly used for lowering cholesterol, has demonstrated promising bone regenerative capability. The aim of this study was to evaluate simvastatin loaded chitosan nanofiber membranes for guided bone regeneration (GBR) applications and their ability to enhance bone formation in rat calvarial defects. Nanofibrous chitosan membranes with random fiber orientation were fabricated by electrospinning technique and loaded with 0.25 mg of simvastatin under sterile conditions. One membrane was implanted subperiosteally to cover an 8 mm diameter critical size calvarial defect. Two groups: 1) Control: non-loaded chitosan membranes;2) Experimental: chitosan membranes loaded with 0.25 mg of simvastatin were evaluated histologically and via micro-computed tomography (micro-CT) for bone formation at 4 and 8 weeks time points (n = 5/group per time point). Both groups exhibited good biocompatibility with only mild or moderate inflammatory response during the healing process. Histologic and micro-CT evaluations confirmed bone formation in calvarial defects as early as 4 weeks using control and experimental membranes. In addition, newly-formed bony bridges consolidating calvarial defects histologically along with partial radiographic defect coverage were observed at 8 weeks in both groups. Although control and experimental groups demonstrated no significant statistical differences in results of bone formation, biodegradable chitosan nanofiber membranes loaded with simvastatin showed a promising regenerative potential as a barrier material for guided bone regeneration applications.

Repair of critical size rat calvarial defects using endometrial-derived stem cells embedded within gelatin/apatite nanocomposite scaffold

Human stromal stem cells derived from endometrium (hESCs) are a type of multipotent stromal cells of the proven ability to differentiate into osteogenic lineage. Thus, it was suggested that these cells may be used to repair skeletal defects. In this study, Human ESCs were extracted from female endometrium and harvested. Biomimetic gelatin/apatite (Gel/Ap) scaffolds with and without harvested cells were implanted in a Critical size calvarial defects in the cranial bone of adult male rat. To CT-Scan and Histological studies were performed to investigate the level of bone formation after 8 weeks of surgery. Results confirmed that the treated defects with the bare and hESCs grafted Gel/Ap scaffold showed significant bone formation and maturation in comparison with the control group.

Tissue Engineered Osteogenesis in Bone Defects by Homologous Osteoblasts Loaded on Sterile Bioresorbable Coral Scaffold in Rabbits

Objectives: This study explores feasibility of tissue-engineered osteogenesis using sterile coral implants loaded with homologous osteoblasts to repair bone defects. Study Design: A unilateral 4 mm transverse dis- continuity defect was produced approximately mid-way along left radius of young female rabbits using ro- tary diamond disc under continuous saline irrigation and stabilised with autoclaved steel miniplate and screws. The defect was then fitted with sterile bioresorbable coral implant loaded with homologous neonatal calvarial osteoblasts or control implants without osteoblasts. All animals underwent radiography immedi- ately post-operative, at weekly intervals for four weeks and at fortnightly intervals thereafter. Operated bones were histologically evaluated for osteogenesis at 12 weeks. Results: Findings demonstrate osteogenesis and complete repair of bioresorbable coral implant by homologous osteoblasts loaded on coral scaffold. Conclu- sions: Single stage surgery using this technique to induce osteogenesis and closure of discontinuity bone de- fects including palatal clefts and peripheral reduction of large craniofacial defects might prove better thera- peutic modality than autologous bone grafting or tissue distraction osteogenesis.

Additively manufactured pure zinc porous scaffolds for critical-sized bone defects of rabbit femur

Additive manufacturing has received attention for the fabrication of medical implants that have customized and complicated structures.Biodegradable Zn metals are revolutionary materials for orthopedic implants.In this study,pure Zn porous scaffolds with diamond structures were fabricated using customized laser powder bed fusion(L-PBF)technology.First,the mechanical properties,corrosion behavior,and biocompatibility of the pure Zn porous scaffolds were characterized in vitro.The scaffolds were then implanted into the rabbit femur critical-size bone defect model for 24 weeks.The results showed that the pure Zn porous scaffolds had compressive strength and rigidity comparable to those of cancellous bone,as well as relatively suitable degradation rates for bone regeneration.A benign host response was observed using hematoxylin and eosin(HE)staining of the heart,liver,spleen,lungs,and kidneys.Moreover,the pure Zn porous scaffold showed good biocompatibility and osteogenic promotion ability in vivo.This study showed that pure Zn porous scaffolds with customized structures fabricated using L-PBF represent a promising biodegradable solution for treating large bone defects.

Biodegradable ZnLiCa ternary alloys for critical-sized bone defect regeneration at load-bearing sites:In vitro and in vivo studies

A novel biodegradable metal system,ZnLiCa ternary alloys,were systematically investigated both in vitro and in vivo.The ultimate tensile strength(UTS)of Zn0.8Li0.1Ca alloy reached 567.60±9.56 MPa,which is comparable to pure Ti,one of the most common material used in orthopedics.The elongation of Zn0.8Li0.1Ca is 27.82±18.35%,which is the highest among the ZnLiCa alloys.The in vitro degradation rate of Zn0.8Li0.1Ca alloy in simulated body fluid(SBF)showed significant acceleration than that of pure Zn.CCK-8 tests and hemocompatibility tests manifested that ZnLiCa alloys exhibit good biocompatibility.Real-time PCR showed that Zn0.8Li0.1Ca alloy successfully stimulated the expressions of osteogenesis-related genes(ALP,COL-1,OCN and Runx-2),especially the OCN.An in vivo implantation was conducted in the radius of New Zealand rabbits for 24 weeks,aiming to treat the bone defects.The Micro-CT and histological evaluations proved that the regeneration of bone defect was faster within the Zn0.8Li0.1Ca alloy scaffold than the pure Ti scaffold.Zn0.8Li0.1Ca alloy showed great potential to be applied in orthopedics,especially in the load-bearing sites.

Irisin-loaded electrospun core-shell nanofibers as calvarial periosteum accelerate vascularized bone regeneration by activating the mitochondrial SIRT3 pathway

The scarcity of native periosteum poses a significant clinical barrier in the repair of critical-sized bone defects.The challenge of enhancing regenerative potential in bone healing is further compounded by oxidative stress at the fracture site.However,the introduction of artificial periosteum has demonstrated its ability to promote bone regeneration through the provision of appropriate mechanical support and controlled release of proosteogenic factors.In this study,a poly(L-lactic acid)(PLLA)/hyaluronic acid(HA)-based nanofibrous membrane was fabricated using the coaxial electrospinning technique.The incorporation of irisin into the core-shell structure of PLLA/HA nanofibers(PLLA/HA@Irisin)achieved its sustained release.In vitro experiments demonstrated that the PLLA/HA@Irisin membranes exhibited favorable biocompatibility.The osteogenic differentiation of bone marrow mesenchymal stem cells(BMMSCs)was improved by PLLA/HA@Irisin,as evidenced by a significant increase in alkaline phosphatase activity and matrix mineralization.Mechanistically,PLLA/HA@Irisin significantly enhanced the mitochondrial function of BMMSCs via the activation of the sirtuin 3 antioxidant pathway.To assess the therapeutic effectiveness,PLLA/HA@Irisin membranes were implanted in situ into critical-sized calvarial defects in rats.The results at 4 and 8 weeks post-surgery indicated that the implantation of PLLA/HA@Irisin exhibited superior efficacy in promoting vascularized bone formation,as demonstrated by the enhancement of bone matrix synthesis and the development of new blood vessels.The results of our study indicate that the electrospun PLLA/HA@Irisin nanofibers possess characteristics of a biomimetic periosteum,showing potential for effectively treating critical-sized bone defects by improving the mitochondrial function and maintaining redox homeostasis of BMMSCs.

Enhancement of critical-sized bone defect regeneration by magnesium oxide-reinforced 3D scaffold with improved osteogenic and angiogenic properties

The healing of critical-sized bone defects(CSD)remains a challenge in orthopedic medicine.In recent years,scaffolds with sophisticated microstructures fabricated by the emerging three-dimensional(3D)printing technology have lighted up the treatment of the CSD due to the elaborate microenvironments and support they may build.Here,we established a magnesium oxide-reinforced 3D-printed biocompos-ite scaffold to investigate the effect of magnesium-enriched 3D microenvironment on CSD repairing.The composite was prepared using a biodegradable polymer matrix,polycaprolactone(PCL),and the disper-sion phase,magnesium oxide(MgO).With the appropriate surface treatment by saline coupling agent,the MgO dispersed homogeneously in the polymer matrix,leading to enhanced mechanical performance and steady release of magnesium ion(Mg^(2+))for superior cytocompatibility,higher cell viability,advanced osteogenic differentiation,and cell mineralization capabilities in comparison with the pure PCL.The in-vivo femoral implantation and critical-sized cranial bone defect studies demonstrated the importance of the 3D magnesium microenvironment,as a scaffold that released appropriate Mg^(2+) exhibited remarkably increased bone volume,enhanced angiogenesis,and almost recovered CSD after 8-week implantation.Overall,this study suggests that the magnesium-enriched 3D scaffold is a potential candidate for the treatment of CSD in a cell-free therapeutic approach.

Calvarial defects and Dandy-Walker malformation in association withneurofibromatosis type 1

Neurofibromatosis type 1(NF1)is an autosomal dominant disorder in which the nerve tissue grows tumors that may be benign and may cause serious damage by compressing nerves and other tissues.The NF1 gene

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute

The development of injectable bone substitutes(IBS)have obtained great importance in the bone re-generation field,as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies.In this scenario,the association of injectable hydrogels and bone graft granules is emerging as a well-established trend.Particularly,in situ forming hydrogels have arisen as a new IBS generation.An in situ forming and injectable dextrin-based hydrogel(HG)was developed,aiming to act as a carrier of granular bone substitutes and bioactive agents.In this work,the HG was associated to a granular bone substitute(Bonelike)and implanted in goat critical-sized calvarial defects(14mm)for 3,6 and 12weeks.The results showed that HG improved the han-dling properties of the Bonelike granules and did not affect its osteoconductive features,neither impairing the bone regener ation process.Human multipotent mesenchymal stromal cells from the umbilical cord,extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS.These bioactive agents did not enhance the new bone formation significantly under the conditions tested,according to micro-computed tomography and histological analysis.

组织工程材料大鼠颅骨缺损修复过程中的磨片三色染色观察

目的:用自体细胞复合含孔磷酸钙陶瓷修复颅骨极量骨缺损的SD大鼠骨组织工程模型标本,制作不脱钙磨片并通过改良Masson氏三色染色以观察新骨形成及其与支架材料的关系。方法:成年雄性SD大鼠 14只,随机分成实验组和对照组,每组 7只。全麻及无菌条件下取其左侧胫、股骨骨髓,体外诱导培养大鼠自体骨髓基质细胞。实验组将已分化的自体成骨细胞与含孔磷酸钙陶瓷复合用于修复同体大鼠颅骨极量骨缺损;对照组颅骨缺损处仅置入无细胞的单纯陶瓷。分别于术后 4、8、12、16、20、24及 28周每组各处死1只动物并取材,制成不脱钙磨片并进行改良Masson氏三色染色,光学显微镜下观察比较颅骨修复情况及其矿化程度。结果:实验组 4周时不脱钙磨片,即可见骨缺损修复中央区陶瓷孔隙内蓝色的新骨形成;随着时间的推移,形成新骨逐渐矿化成为红色的成熟骨组织, 28周时骨缺损修复区可见大片新骨呈岛状或条索状排列,其间的陶瓷支架部分已降解,同时加以墨汁灌注的磨片显示骨组织中已形成大量新生血管网。结论:以含孔磷酸钙陶瓷为支架的组织工程骨缺损修复明显优于单纯生物陶瓷修复法,磨片三色染色方法既可了解新骨形成及其矿化程度,又可观察新骨形成与陶瓷支架之间的关系。

海藻酸钠壳聚糖支架复合成骨细胞特异性多肽修复兔颅骨缺损

目的评价成骨细胞特异性识别多肽在骨缺损中的修复效果。方法将成骨细胞特异性识别多肽与海藻酸钠/壳聚糖水凝胶(SA/CS Gel)复合植入兔颅骨双侧15 mm圆形极限骨缺损区,实验组缺损处植入海藻酸钠/壳聚糖/成骨细胞特异性识别多肽水凝胶(SA/CS/多肽Gel),对照组植入SA/CS Gel,空白组不植入任何载体及药物,随机分组。术后8周活体行CBCT扫描,随后处死取材并常规HE染色行组织学检查观察成骨效果。结果影像学及组织形态学结果可见,实验组缺损区成骨量明显大于对照组及空白组,差异有统计学意义(P<0.05)。结论成骨细胞特异性识别多肽可促进兔颅骨缺损的骨修复。

组织工程颅骨缺损修复过程中超微结构观察

目的 用组织工程骨修复 SD大鼠颅骨极量骨缺损 ,并在透射电镜下观察骨修复过程中成骨细胞、血管内皮细胞与陶瓷支架的关系。方法 14只雄性 SD大鼠 ,随机分成实验组和对照组 ,每组 7只。取左侧胫、股骨骨髓 ,体外诱导培养骨髓基质细胞。实验组将已分化的成骨细胞与含孔磷酸钙陶瓷复合用于修复大鼠自体颅骨极量骨缺损 ;对照组颅骨缺损处仅置入无细胞的陶瓷材料。分别于术后 4、8、12、16、2 0、2 4及 2 8周每组各处死 1只动物取材 ,制成脱钙超薄切片 ,透射电镜下观察成骨细胞、血管内皮细胞与陶瓷残余支架的关系。结果 实验组动物颅骨修复 12周前成骨细胞或成骨细胞样细胞毗邻新生小血管、血管内皮细胞存在 ,16周后成骨细胞转变为骨细胞 ,并围绕血管被包埋于骨基质及胶原纤维中 ;陶瓷残余与新生骨间有一条明显的钙化沉积带 ,大量胶原纤维已伸入到陶瓷支架的纳米级结构中。对照组陶瓷中央部位有大量小血管及血管内皮细胞增生 ,其周围多为幼稚成纤维细胞 ,无成骨细胞与血管内皮细胞的依附关系。结论 新型含孔磷酸钙陶瓷作为组织工程细胞支架 ,其间的纳米级结构有利于骨的再生和血管形成 ;未见来源于植骨床血管的内皮细胞衍变为成骨细胞。

Mimics与Micro-CT内置软件测量小鼠颅骨缺损骨再生的对比研究

目的:对比分析Mimics软件与显微-CT(Micro-computed tomography,Micro-CT)内置分析软件对颅骨缺损内新生骨量及骨愈合率的测量结果,评价2种测量方法的差异性。方法:在小鼠右侧颅顶骨制备直径4 mm圆形骨缺损,分别于术后当天、6周、12周,对小鼠颅骨进行活体Micro-CT扫描,然后利用Micro-CT扫描仪内置软件重建颅骨缺损的三维图像并测量缺损内新生骨体积及愈合率,且利用Mimics软件对Micro-CT扫描的二维图片进行三维重建,并测量缺损内新生骨体积及愈合率。结果:与术后6周相比,术后12周Micro-CT扫描仪内置软件和Mimics软件测量的缺损内新生骨体积和骨愈合率均增加,差异有统计学意义(P<0.05);术后6周和12周,Mimics软件的测量结果与Micro-CT扫描仪内置软件测量的颅骨缺损内新骨体积及骨愈合率相比,差异无统计学意义(P>0.05)。结论:缺损术后6周和术后12周,Mimics软件对Micro-CT扫描后的二维图片进行重建并计算出的颅骨缺损新生骨体积及骨愈合率,与Micro-CT扫描仪内置软件的测量结果均一致,且测量方法更简单。

骨髓间充质干细胞复合多孔纳米羟磷灰石/聚酰胺6整复大鼠颅骨缺损的实验研究

目的初步探讨纳米羟磷灰石/聚酰胺6(n-HA/PA6)多孔材料对体外培养的大鼠骨髓间充质干细胞(BM-SCs)增殖及分化的影响,以及BMSCs作为种子细胞、多孔n-HA/PA6作为支架材料构建组织工程化骨修复大鼠颅骨极限骨缺损的可行性及整复效果。方法矿化诱导的第3代BMSCs与n-HA/PA6多孔材料复合培养,MTT检测细胞增殖,ALP染色检测骨向分化。将BMSCs与n-HA/PA6复合物植入大鼠颅骨8mm骨缺损处,4、8、16周时,应用组织学、扫描电镜观察植入物与骨组织交界处的成骨修复情况,并与单纯n-HA/PA6植入组修复效果进行比较。结果与n-HA/PA6复合培养的BMSCs生长良好,细胞增殖未受影响,ALP染色阳性。BMSCs复合n-HA/PA6植入4周时,有较多新骨长入支架孔穴;8周时,材料和宿主骨融为一体,接近正常骨;16周时,材料和天然骨形成骨性结合。单纯n-HA/PA6组植入4周时,新骨形成较少;8周时,新骨明显增加,但骨钙化程度较低;16周时,2组无明显差异。结论多孔n-HA/PA6支架材料对种子细胞BMSCs的增殖和骨向分化无影响;BMSCs作为种子细胞、n-HA/PA6多孔复合体作为支架材料构建组织工程化骨能够加速界面骨愈合,有效修复颅骨缺损,具有潜在的骨组织工程应用前景。

鸵鸟骨转化羟基磷灰石支架复合骨膜修复颅骨极限缺损实验研究

目的:观察鸵鸟骨转化羟基磷灰石支架复合骨膜修复颅骨缺损的成骨性能。方法:鸵鸟骨转化多孔羟基磷灰石作为支架材料。24只新西兰大白兔随机分为3组并在其颅骨上制备极限缺损,修复方法分别为A组:支架材料+骨膜植入,B组:单纯支架材料植入,C组:自体颅骨植入。植入后8、16周取材,通过X线片分析、大体和组织学观察评价其成骨性能。结果:大体观察发现A组陶瓷材料孔隙表面及内部有大量新骨形成,新骨与周围颅骨发生骨性融合,其强度与邻近颅骨接近。X-线片显示A组绝大多数区域呈现高密度阻射影。组织学观察显示新的板层状骨组织、骨髓腔及骨髓细胞在陶瓷支架孔隙表面及内部形成。B组材料大部分被纤维组织覆盖或充填。结论:支架/骨膜复合物能够有效地修复颅骨缺损,结果提示鸵鸟骨转化羟基磷灰石能够对骨膜提供良好的支持作用。

细胞-膜片复合珊瑚支架修复兔颅骨极限缺损的实验研究

目的:评价富血小板血浆(platelet rich plasma,PRP)和骨髓基质细胞(marrow stromal cells,MSCs)构建的细胞-膜片包裹珊瑚支架后修复兔颅骨极限缺损的能力。方法:将体外培养扩增的兔MSCs悬液与同一供体来源的PRP混合,滴加到六孔培养板上,再滴加牛凝血酶溶液,形成凝胶样MSCs/PRP混合物,置于孵箱内培养。第1周用DMEM完全培养液,第2周用DMEM诱导培养液培养,形成细胞-膜片。将细胞-膜片取下后包裹珊瑚支架,植入供体兔颅骨直径15 mm的圆形缺损内,用自体颅骨和单纯珊瑚植入作对照。术后8周和16周取材,通过大体观察、组织学观察和组织形态测量分析等,评价其骨修复效果。结果:培养的细胞-膜片厚约2 mm,呈半透明胶冻样,有较好的韧性和可操作性。细胞-膜片/珊瑚组修复兔颅骨缺损效果明显优于单纯珊瑚修复组,在16周时得到完全的骨修复,与自体骨类似。结论:用MSCs和PRP在体外培养构建的细胞-膜片具有较强的成骨活性,包裹珊瑚支架后具有良好的骨修复能力。