目的：骨缺损一直是困扰临床医生的难题之一，随着骨组织工程的不断发展，涌现出大量的生物材料：拥有良好的生物相容性、优秀的弹性模量，但仍很难应用到临床中，其原因是这些材料内部缺乏成骨必要的细胞或成骨因子等，造成骨长入量较低，仅在生物材料表面有少量骨生成，而骨组织很难长入材料的内部。因此，我们拟利用可吸收凝胶加载间充质干细胞及其外泌体，分别在体外检测其促骨髓间充质干细胞成骨的效果，并在动物骨缺损模型中验证其体内骨修复的效果，以探寻可吸收凝胶装载间充质干细胞或其外泌体促成骨的可行性，为临床修复骨缺损提供可靠的理论依据。

方法：1.首先通过光镜观察间充质干细胞的形态、大小、密度等变化，并利用流式细胞仪检测BMSCs表达的特异性分子标志物；2.将BMSCs包裹于水凝胶中，比较在二维和三维培养环境下BMSCs形态、大小、密度、增殖能力及水凝胶与细胞间的生物相容性。3.利用茜素红染色和碱性磷酸酶活性检测方法观察三维培养条件下BMSCs向成骨细胞定向分化的情况，再通过Western blotting和qRT-PCR检测成骨蛋白和基因的表达情况。4.制备SD大鼠全层下颌骨缺损的动物模型，分为四组：单纯钛合金支架组（BS组），支架+注射细胞组（SC组），支架+凝胶组（SM组）和支架+凝胶+细胞组（SMC组）分别修复大鼠的下颌骨缺损，在6周和12周时，通过micro-CT、硬组织切片及VG染色来评价骨缺损修复的效果。5.使用超速离心法分离、提纯UMSCs在普通培养和成骨诱导培养条件下的外泌体（分别为UMSC-Exo，UMSC-OI-Exo），并对提取的外泌体进行鉴定。6.分为三组进行体内及体外实验：无外泌体（control组）；普通外泌体（UMSC-Exo组）；成骨诱导来源外泌体（UMSC-OI-Exo组）。利用CCK8及划痕实验检测两种外泌体对BMSCs增殖及迁移能力的影响。7.再利用茜素红和碱性磷酸酶染色方法检测两种外泌体对BMSCs成骨能力的影响；使用Western blotting和qRT-PCR验证成骨相关蛋白和mRNA表达水平。8.合成粘附力较强的透明质酸-多巴胺（HA-CA）凝胶作为外泌体的载体，分别检测HA-CA对于外泌体的保留能力及在凝胶内BMSCs对于外泌体的内吞效果。9.按照前述方法制备大鼠下颌骨缺损模型，并用HA-CA凝胶加载外泌体对骨缺损进行修复，共分三组：单纯凝胶组（HA-CA组），普通UMSCs外泌体组（UMSC-Exo组）和UMSCs成骨外泌体组（UMSC-OI-Exo组）。通过micro-CT及组织病理学染色（HE、Masson方法）评价三组对于骨缺损的修复效果。10.利用转录组测序技术找寻两种外泌体在miRNA水平差异，并通过生信分析找寻其下游靶基因，探寻UMSC-OI-Exo促成骨的潜在机制。

结果：1.细胞在培养24h后呈纺锤状或成纤维细胞样，伪足明显、簇状生长，具有良好的成骨分化潜能；BMSCs表达抗原：CD44、CD73、CD90、CD105、HLA-ABC呈强阳性表达，阳性率分别为98.65%，99.73%，95.69%，97.32%，99.87%。2.我们成功的将BMSCs包裹于Matrigel凝胶中，光镜下观察细胞呈三维方式生长，细胞间连接更紧密；BMSCs在三维培养条件下可稳定增殖，且活细胞数可达95%以上。3.BMSCs在水凝胶中三维培养时，茜素红染色及碱性磷酸酶活性明显高于二维培养体系；且成骨相关蛋白及mRNA的表达也高于二维培养。4.动物实验发现：SMC组生成的骨组织更加致密，在12周时支架内部有大量的新生骨组织覆盖；硬组织切片及VG染色结果与micro-CT结果一致：BS组和SC组有大量纤维组织，但几乎没有骨组织长入，而SMC组支架内生成大量的骨组织。5.电镜检测两种外泌体均具有脂质双层膜结构，表达外泌体特异性标志物。6.两种外泌体均可促进BMSCs增殖，其中UMSCs-OI-Exo可以加速BMSCs的迁移。7.茜素红和碱性磷酸酶染色发现UMSCs-OI-Exo可以明显增加BMSCs的矿化，且UMSCs-OI-Exo组中成骨相关蛋白和mRNA的表达均高于其它两组。8.HA-CA凝胶内部呈现规则的中空网状结构，孔径大约在30-50μm之间，能够很好的保留外泌体，并且两种外泌体均可被BMSCs内吞入细胞中。9.Micro-CT扫描发现HA-CA组在12周时仍无骨缺损修复迹象，UMSC-Exo组在12周时才有少量成骨，而UMSC-OI-Exo组在6周时就出现部分骨组织，在12周时骨缺损中心骨量明显增加，通过H&E及Masson染色进一步证实了这一结果。10.miRNA测序结果显示，UMSC-OI-Exo中高表达miR-199a-3p,可能通过与其靶基因mTOR结合从而提高BMSCs的成骨效果。

结论：1.BMSCs生长情况良好，符合干细胞特性，且具有良好的定向分化潜力，为后续研究提供种子细胞。2.BMSCs包裹于水凝胶中不影响细胞的增殖，并能够促进细胞间的连接，为进一步成骨分化实验提供良好的研究基础。3. BMSCs在水凝胶中的三维培养可明显增强其成骨能力，并在体外定向分化为成骨细胞，从而加速体内骨缺损的修复，此方法优于BMSCs单纯注射或单纯水凝胶植入法。4.超速离心法能够成功提取间充质干细胞外泌体，且两种外泌体（UMSC-Exo，UMSC-OI-Exo）在形态上无差异。5.通过体外实验证实UMSCs-OI-Exo能显著增强BMSCs的活性，并提升后者的成骨能力。6.体内实验证实可吸收凝胶HA-CA载UMSC-OI-Exo够有效修复下颌骨缺损。7.其中miR-199a-3p在调节BMSCs成骨过程中可能扮演了重要角色。本研究结果可为临床医生治疗骨缺损提供了新的方法，具有重要的临床意义。

ive: bone defect is one of the problems that plague clinicians. with the development of bone tissue engineering, a large number of biological materials have emerged, with good biocompatibility and excellent elastic modulus. however, there is still a long way of clinical application. the reason is that there is no osteogenic cells or factors inside these biomaterials, which lead to low bone growth. there is only a little bone formation on the surface of the biological materials, but no bone tissue growing inside of the biomaterials. therefore, we loaded mesenchymal stem cells (mscs) or mscs-derived exosomes into absorbable hydrogels, and tested their osteogenic differentiation capacity in vitro, and also verify the bone repair ability in vivo in the bone defect rat model. we would like to explore the feasibility of absorbable hydrogels loaded mscs or their-derived exosomes of promoting osteogenic ability, and providing a reliable theory for clinical repair of bone defects.

methods: 1. during the cultural process, the morphology, size, density and osteogenic potentials of the cells were observed by microscopes and the specific molecular markers of bmscs were also detected by flow cytometry. 2. bmscs were encapsulated in hydrogel to compare the cell morphology, size, density, proliferation ability and biocompatibility of hydrogel between two-dimensional and three-dimensional cultural environments. 3. we observed the osteogenic differentiation of bmscs through alizarin red staining and alp activity. then, the expression of osteogenic proteins and mrnas were detected through western blotting and qrt-pcr. 4. animal models of full-thickness mandibular defects in sd rats were prepared and divided into four groups: bare scaffold (bs group), scaffold with injected bmscs + pbs (sc group), scaffold + matrigel (sm group) and scaffold + bmsc-loaded matrigel (smc group) were used to repair the mandibular defects. micro-ct and histological analysis (van-gieson staining) were used to characterize mineralized formation of all defects at 6 and 12 weeks. 5. we purified and identified exosomes from umscs cultured with mscs media or osteogenic induction media, respectively (named as umsc-exo, umsc-oi-exo). 6. three groups were used to perform in vitro experiments: no exosomes (control group), umscs derived exosomes with mscs media (umsc-exo group) and umscs derived exosomes with osteogenic induced media (umsc-oi-exo group). then, cck8 and scratch assay were used to detect the proliferation and migration effect of bmscs among the three groups. 7 alizarin red and alkaline phosphatase staining were used to detect the osteogenesis of bmscs. we also examined the expression of osteogenic protein and mrna by western blotting and qrt-pcr. 8. an adhesion absorbable hydrogel hyaluronic acid-catechol (ha-ca) was synthesized as a carrier for exosomes. the retention ability of ha-ca for exosomes and the endocytosis of exosomes by bmscs was also detected. 9. the animal model was prepared as pervious described and divided into three groups: hydrogel (ha-ca group), umscs basic exosomes (umsc-exo group) and umscs osteogenic exosomes (umsc-oi-exo group). the therapeutic effect was evaluated by micro-ct and histopathological staining (he and masson staining). 10. we used mirna sequencing to compare the different expression of mirna between two exosomes and to search for mirna networks through gene ontology and kegg to explore the potential mechanisms that contribute to bone regeneration.

results: 1. after culturing for 24 hours, the cells were spindle-shaped with obvious pseudopods, cluster-like growth, and had osteogenic differentiation potential. the expression of bmscs surface antigen was found: cd44, cd73, cd90, cd105, hla-abc strong positive and the positive rates were 98.65%, 99.73%, 95.69%, 97.32%, 99.87%, respectively; 2. bmscs were successfully wrapped in matrigel, and bmscs can grow stably in a three-dimensional environment and have more cell connection, with more than 95% living cells. 3. bmscs cultured in three-dimensional environment have significantly higher mineralized deposition in alizarin red staining and higher alp activity. in western blotting and qrt-pcr experiments, the expression of osteogenic proteins and mrnas were higher in 3d environment than 2d. 4. in vivo experiments also showed that the regeneration of bone tissue in the smc group was denser and there was large amount of new bone inside the scaffolds after 12 weeks of implantation. the histological analysis showed that there were many fibrous tissues inside the bs group and sc group, but rare bone tissue. however, there were many bone tissue grown in the smc group, which were tightly bound to the scaffolds. 5. both exosomes had typical double-membranes structure and expressed specific marker. 6. both exosomes could promote the proliferation of bmscs. and umscs-oi-exo significantly increased the migration rate of bmscs. 7. alizarin red and alkaline phosphatase staining showed that umscs-oi-exo significantly increased the mineralization of bmscs. western blotting and qrt-pcr experiments also showed that the expression of osteogenic proteins and mrnas were highest in umscs-oi-exo group. 8. the ha-ca hydrogel showed a highly porous network structure with 30-50 μm in diameter, which can retain exosomes. and two kinds of exosomes could be internalized by bmscs within ha-ca hydrogel. 9. micro-ct scan showed that there was no obvious bone regeneration in ha-ca group and only minor bone regeneration in umsc-exo group at 12 weeks. while there was large amount of new bone generated in umsc-oi-exo group at 12 weeks. and h&e and masson staining further supported these results. 10. mirna sequencing showed that mir-199a-3p was highly enriched in umsc-oi-exo, which may improve the osteogenic effect of mesenchymal stem cells by inhibiting target gene mtor.

conclusions: 1. the bmscs grown well and matched stem cell characteristics, with good potential of differentiation, which can be used as seed cells for subsequent experiments. 2.the encapsulation of bmscs in hydrogels does not affect the cell proliferation potential, and can also improve cell-to-cell connection.3.3d cultured bmscs can significantly enhance osteogenic capacity, both in vivo and in vitro, thereby, accelerating the healing of bone defects. this method was superior to the simple bmscs injection or the hydrogel implantation methods. 4. ultracentrifugation can successfully extract exosomes and there is no difference in morphology between the two kinds of exosomes. 5. and the umscs-oi-exo could enhance the biological activity and osteogenic ability of bmscs. 6. umsc-oi-exo combined with absorbable hydrogel (ha-ca) can effectively repair bone defects. 7.we found that mir-199a-3p may plays an important role in regulating the osteogenesis of bmscs. the results of this study can provide a new method for treating bone defects clinically, which has important clinical significance.