Exosomes: A New Hope for Angiogenesis-Mediated Bone Regeneration.

Bone is a metabolically dynamic structure that is generally remodeled throughout the lifetime of an individual but often causes problems with increasing age. A key player for bone development and homeostasis, but also under pathological conditions, is the bone vasculature. This complex system of arteries, veins, and capillaries forms distinct structures where each subset of endothelial cells has important functions. Starting with the basic process of angiogenesis and bone-specific blood vessel formation, coupled with initial bone formation, the importance of different vascular structures is highlighted with respect to how these structures are maintained or changed during homeostasis, aging, and pathological conditions. After exemplifying the current knowledge on bone vasculature, this review will move on to exosomes, a novel hotspot of scientific research. Exosomes will be introduced starting from their discovery via current isolation procedures and state-of-the-art characterization to their role in bone vascular development, homeostasis, and bone regeneration and repair while summarizing the underlying signal transduction pathways. With respect to their role in these processes, especially mesenchymal stem cell-derived extracellular vesicles are of interest, which leads to a discussion on patented applications and an update on ongoing clinical trials. Taken together, this review provides an overview of bone vasculature and bone regeneration, with a major focus on how exosomes influence this intricate system, as they might be useful for therapeutic purposes in the near future.

Clinical potential of plasma-functionalized graphene oxide ultrathin sheets for bone and blood vessel regeneration: Insights from cellular and animal models.

Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.

Calcitonin gene-related peptide-modulated macrophage phenotypic alteration regulates angiogenesis in early bone healing.

OBJECTIVES: This study aimed to investigate the effect of calcitonin gene-related peptide (CGRP) on the temporal alteration of macrophage phenotypes and macrophage-regulated angiogenesis duringearlybonehealing and preliminarily elucidate the mechanism. METHODS: In vivo, the rat mandibular defect models were established with inferior alveolar nerve transection (IANT) or CGRP receptor antagonist injection. Radiographicandhistologic assessments for osteogenesis, angiogenesis, and macrophage phenotypic alteration within bone defects were performed. In vitro, the effect and mechanism of CGRP on macrophage polarization and phenotypic alteration were analyzed. Then the conditioned medium (CM) from CGRP-treated M1 or M2 macrophages was used to culture human umbilical vein endothelial cells (HUVECs), and the CGRP's effect on macrophage-regulated angiogenesis was detected. RESULTS: Comparable changes following IANT and CGRP blockade within bone defects were observed, including the suppression of early osteogenesis and angiogenesis, the prolonged M1 macrophage infiltration and the prohibited transition toward M2 macrophages around vascular endothelium. In vitro experiments showed that CGRP promoted M2 macrophage polarization while upregulating the expression of interleukin 6 (IL-6), a major cytokine that facilitates the transition from M1 to M2-dominant stage, in M1 macrophages via the activation of Yes-associated protein 1. Moreover, CGRP-treated macrophage-CM showed an anabolic effect on HUVECs angiogenesis compared with macrophage-CM and might prevail over the direct effect of CGRP on HUVECs. CONCLUSIONS: Collectively, our results reveal the effect of CGRP on M1 to M2 macrophage phenotypic alteration possibly via upregulating IL-6 in M1 macrophages, and demonstrate the macrophage-regulated pro-angiogenic potential of CGRP in early bone healing.

Fabrication of vascularized tissue-engineered bone models using triaxial bioprinting.

Bone tissue is a highly vascularized tissue. When constructing tissue-engineered bone models, both the osteogenic and angiogenic capabilities of the construct should be carefully considered. However, fabricating a vascularized tissue-engineered bone to promote vascular formation and bone generation, while simultaneously establishing nutrition channels to facilitate nutrient exchange within the constructs, remains a significant challenge. Triaxial bioprinting, which not only allows the independent encapsulation of different cell types while simultaneously forming nutrient channels, could potentially emerge as a strategy for fabricating vascularized tissue-engineered bone. Moreover, bioinks should also be applied in combination to promote both osteogenesis and angiogenesis. In this study, employing triaxial bioprinting, we used a blend bioink of gelatin methacryloyl (GelMA), sodium alginate (Alg), and different concentrations of nano beta-tricalcium phosphate (nano ß-TCP) encapsulated MC3T3-E1 preosteoblasts as the outer layer, a mixed bioink of GelMA and Alg loaded with human umbilical vein endothelial cells (HUVEC) as the middle layer, and gelatin as a sacrificial material to form nutrient channels in the inner layer to fabricate vascularized bone constructs simulating the microenvironment for bone and vascular tissues. The results showed that the addition of nano ß-TCP could adjust the mechanical, swelling, and degradation properties of the constructs. Biological assessments revealed the cell viability of constructs containing different concentrations of nano ß-TCP was higher than 90% on day 7, The cell-laden constructs containing 3% (w/v) nano ß-TCP exhibited better osteogenic (higher Alkaline phosphatase activity and larger Osteocalcin positive area) and angiogenic (the gradual increased CD31 positive area) potential. Therefore, using triaxial bioprinting technology and employing GelMA, Alg, and nano ß-TCP as bioink components could fabricate vascularized bone tissue constructs, offering a novel strategy for vascularized bone tissue engineering.

Hydrogel-Based Systems in Neuro-Vascularized Bone Regeneration: A Promising Therapeutic Strategy.

Blood vessels and nerve fibers are distributed throughout the skeletal tissue, which enhance the development and function of each other and have an irreplaceable role in bone formation and remodeling. Despite significant progress in bone tissue engineering, the inadequacy of nerve-vascular network reconstruction remains a major limitation. This is partly due to the difficulty of integrating and regulating multiple tissue types with artificial materials. Thus, understanding the anatomy and underlying coupling mechanisms of blood vessels and nerve fibers within bone to further develop neuro-vascularized bone implant biomaterials is an extremely critical aspect in the field of bone regeneration. Hydrogels have good biocompatibility, controllable mechanical characteristics, and osteoconductive and osteoinductive properties, making them important candidates for research related to neuro-vascularized bone regeneration. This review reports the classification and physicochemical properties of hydrogels, with a focus on the application advantages and status of hydrogels for bone regeneration. The authors also highlight the effect of neurovascular coupling on bone repair and regeneration and the necessity of achieving neuro-vascularized bone regeneration. Finally, the recent progress and design strategies of hydrogel-based biomaterials for neuro-vascularized bone regeneration are discussed.

Type H blood vessels in coupling angiogenesis-osteogenesis and its application in bone tissue engineering.

One specific capillary subtype, termed type H vessel, has been found with unique functional characteristics in coupling angiogenesis with osteogenesis. Researchers have fabricated a variety of tissue engineering scaffolds to enhance bone healing and regeneration through the accumulation of type H vessels. However, only a limited number of reviews discussed the tissue engineering strategies for type H vessel regulation. The object of this review is to summary the current utilizes of bone tissue engineering to regulate type H vessels through various signal pathways including Notch, PDGF-BB, Slit3, HIF-1α, and VEGF signaling. Moreover, we give an insightful overview of recent research progress about the morphological, spatial and age-dependent characteristics of type H blood vessels. Their unique role in tying angiogenesis and osteogenesis together via blood flow, cellular microenvironment, immune system and nervous system are also summarized. This review article would provide an insight into the combination of tissue engineering scaffolds with type H vessels and identify future perspectives for vasculized tissue engineering research.

Effect of bone morphogenic protein-2 and desferoxamine on distraction osteogenesis.

BACKGROUND: Angiogenesis is crucial for formation of a stable regenerate during distraction osteogenesis (DO). This experimental study evaluates if bone morphogenic protein-2 (BMP-2) and desferrioxamine (DFO), two agents which are known to induce neoangiogenesis in vivo, would increase angiogenesis and osteogenesis, and improve mechanical properties of bone regenerate in DO model. METHODS: Twenty-four tibias of 24 New Zealand rabbits were osteotomized and fixed with semi-circular fixators. Three groups of 8 animals were formed. BMP-2 soaked scaffolds were used in the first group, whereas daily local DFO injections were made in the second group. Subjects in the control group did not receive any agents during the surgery or in the distraction period. The rabbits in all three groups underwent distraction at a rate of 0.6 mm/day for 15 days following the 7-day latent period. Animals were sacrificed on day 38, and the tibia were harvested for histological and mechanical examination of the regenerate. RESULTS: All 24 rabbits survived the surgical procedure, and there were no side effects against the BMP-2 and local DFO. Three-point bending tests revealed a higher force (361 ± 267 N.) required for fracture in Group 1 (p: 0.018). Similarly, the bending moment in Group 1 (5.4 ± 4.0 Nmm) was significantly higher than the other groups (p: 0.021). There was no significant difference between the groups in terms of deflection and stiffness (p Ëƒ 0.05). Histologically, there was no statistical difference between the groups in terms of endochondral, periosteal, and intramembranous ossification and VEGF activity (p Ëƒ 0.05). CONCLUSION: BMP-2 and DFO stimulate angiogenesis by increasing VEGF activity. Angiogenesis is one of the most important mechanisms for the initiation and maintenance of new bone formation. Stimulation of angiogenesis in unfavorable biomechanical conditions may not be sufficient for ideal bone formation.

Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model.

Osteopontin (OPN) has been proved to be closely related to the pathogenesis of osteoarthritis (OA), but the role of OPN in the pathogenesis of OA has not been fully clarified. Current studies on OPN in OA mostly focus on articular cartilage, synovial membrane and articular fluid, while ignoring its role in OA subchondral bone turnover and remodeling. In this study, we used a destabilization OA mouse model to investigate the role of OPN in OA subchondral bone changes. Our results indicate that increased expression of OPN accelerates the turnover and remodeling of OA subchondral bone, promotes the formation of h-type vessels in subchondral bone, and mediates articular cartilage degeneration induced by subchondral bone metabolism. In addition, our results confirmed that inhibition of PI3K/AKT signaling pathway inhibits OPN-mediated OA subchondral bone remodeling and cartilage degeneration. This study revealed the role and mechanism of OPN in OA subchondral bone, which is of great significance for exploring specific biological indicators for early diagnosis of OA and monitoring disease progression, as well as for developing drugs to regulate the metabolism and turnover of subchondral bone and alleviate the subchondral bone sclerosis of OA.

Spatiotemporal regulation of angiogenesis/osteogenesis emulating natural bone healing cascade for vascularized bone formation.

Engineering approaches for growth factor delivery have been considerably advanced for tissue regeneration, yet most of them fail to provide a complex combination of signals emulating a natural healing cascade, which substantially limits their clinical successes. Herein, we aimed to emulate the natural bone healing cascades by coupling the processes of angiogenesis and osteogenesis with a hybrid dual growth factor delivery system to achieve vascularized bone formation. Basic fibroblast growth factor (bFGF) was loaded into methacrylate gelatin (GelMA) to mimic angiogenic signalling during the inflammation and soft callus phases of the bone healing process, while bone morphogenetic protein-2 (BMP-2) was bound onto mineral coated microparticles (MCM) to mimics osteogenic signalling in the hard callus and bone remodelling phases. An Initial high concentration of bFGF accompanied by a sustainable release of BMP-2 and inorganic ions was realized to orchestrate well-coupled osteogenic and angiogenic effects for bone regeneration. In vitro experiments indicated that the hybrid hydrogel markedly enhanced the formation of vasculature in human umbilical vein endothelial cells (HUVECs), as well as the osteogenic differentiation of mesenchymal stem cells (BMSCs). In vivo results confirmed the optimal osteogenic performance of our F/G-B/M hydrogel, which was primarily attributed to the FGF-induced vascularization. This research presents a facile and potent alternative for treating bone defects by emulating natural cascades of bone healing.

Human Periodontal Ligament Stem Cell and Umbilical Vein Endothelial Cell Co-Culture to Prevascularize Scaffolds for Angiogenic and Osteogenic Tissue Engineering.

(1) Background: Vascularization remains a critical challenge in bone tissue engineering. The objective of this study was to prevascularize calcium phosphate cement (CPC) scaffold by co-culturing human periodontal ligament stem cells (hPDLSCs) and human umbilical vein endothelial cells (hUVECs) for the first time; (2) Methods: hPDLSCs and/or hUVECs were seeded on CPC scaffolds. Three groups were tested: (i) hUVEC group (hUVECs on CPC); (ii) hPDLSC group (hPDLSCs on CPC); (iii) co-culture group (hPDLSCs + hUVECs on CPC). Osteogenic differentiation, bone mineral synthesis, and microcapillary-like structures were evaluated; (3) Results: Angiogenic gene expressions of co-culture group were 6-9 fold those of monoculture. vWF expression of co-culture group was 3 times lower than hUVEC-monoculture group. Osteogenic expressions of co-culture group were 2-3 folds those of the hPDLSC-monoculture group. ALP activity and bone mineral synthesis of co-culture were much higher than hPDLSC-monoculture group. Co-culture group formed capillary-like structures at 14-21 days. Vessel length and junction numbers increased with time; (4) Conclusions: The hUVECs + hPDLSCs co-culture on CPC scaffold achieved excellent osteogenic and angiogenic capability in vitro for the first time, generating prevascularized networks. The hPDLSCs + hUVECs co-culture had much better osteogenesis and angiogenesis than monoculture. CPC scaffolds prevacularized via hPDLSCs + hUVECs are promising for dental, craniofacial, and orthopedic applications.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division.

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.

The endothelium-bone axis in development, homeostasis and bone and joint disease.

Blood vessels form a versatile transport network that is best known for its critical roles in processes such as tissue oxygenation, metabolism and immune surveillance. The vasculature also provides local, often organ-specific, molecular signals that control the behaviour of other cell types in their vicinity during development, homeostasis and regeneration, and also in disease processes. In the skeletal system, the local vasculature is actively involved in both bone formation and resorption. In addition, blood vessels participate in inflammatory processes and contribute to the pathogenesis of diseases that affect the joints, such as rheumatoid arthritis and osteoarthritis. This Review summarizes the current understanding of the architecture, angiogenic growth and functional properties of the bone vasculature. The effects of ageing and pathological conditions, including arthritis and osteoporosis, are also discussed.

Vascularization Strategies in Bone Tissue Engineering.

Bone is a highly vascularized tissue, and its development, maturation, remodeling, and regeneration are dependent on a tight regulation of blood vessel supply. This condition also has to be taken into consideration in the context of the development of artificial tissue substitutes. In classic tissue engineering, bone-forming cells such as primary osteoblasts or mesenchymal stem cells are introduced into suitable scaffolds and implanted in order to treat critical-size bone defects. However, such tissue substitutes are initially avascular. Because of the occurrence of hypoxic conditions, especially in larger tissue substitutes, this leads to the death of the implanted cells. Therefore, it is necessary to devise vascularization strategies aiming at fast and efficient vascularization of implanted artificial tissues. In this review article, we present and discuss the current vascularization strategies in bone tissue engineering. These are based on the use of angiogenic growth factors, the co-implantation of blood vessel forming cells, the ex vivo microfabrication of blood vessels by means of bioprinting, and surgical methods for creating surgically transferable composite tissues.

Hypertension meets osteoarthritis - revisiting the vascular aetiology hypothesis.

Osteoarthritis (OA) is a whole-joint disease characterized by subchondral bone perfusion abnormalities and neovascular invasion into the synovium and articular cartilage. In addition to local vascular disturbance, mounting evidence suggests a pivotal role for systemic vascular pathology in the aetiology of OA. This Review outlines the current understanding of the close relationship between high blood pressure (hypertension) and OA at the crossroads of epidemiology and molecular biology. As one of the most common comorbidities in patients with OA, hypertension can disrupt joint homeostasis both biophysically and biochemically. High blood pressure can increase intraosseous pressure and cause hypoxia, which in turn triggers subchondral bone and osteochondral junction remodelling. Furthermore, systemic activation of the renin-angiotensin and endothelin systems can affect the Wnt-ß-catenin signalling pathway locally to govern joint disease. The intimate relationship between hypertension and OA indicates that endothelium-targeted strategies, including re-purposed FDA-approved antihypertensive drugs, could be useful in the treatment of OA.

Post-myocardial infarction heart failure dysregulates the bone vascular niche.

The regulation of bone vasculature by chronic diseases, such as heart failure is unknown. Here, we describe the effects of myocardial infarction and post-infarction heart failure on the bone vascular cell composition. We demonstrate an age-independent loss of type H endothelium in heart failure after myocardial infarction in both mice and humans. Using single-cell RNA sequencing, we delineate the transcriptional heterogeneity of human bone marrow endothelium, showing increased expression of inflammatory genes, including IL1B and MYC, in ischemic heart failure. Endothelial-specific overexpression of MYC was sufficient to induce type H bone endothelial cells, whereas inhibition of NLRP3-dependent IL-1ß production partially prevented the post-myocardial infarction loss of type H vasculature in mice. These results provide a rationale for using anti-inflammatory therapies to prevent or reverse the deterioration of bone vascular function in ischemic heart disease.

Histological analysis of induced membranes in patients whose bone defects were treated with the Masquelet technique to identify factors affecting the vascularity of induced membranes.

BACKGROUND: Rich vascularity of the induced membrane (IM) is important for Masquelet reconstruction surgery. The factors affecting IM vascularity are not completely understood. This study aimed to investigate these factors using histological samples of human IMs. METHODS: We retrospectively evaluated 36 patients whose bone defects were treated using the Masquelet technique. Two clinical pathologists analyzed histological sections of IM pieces (1 cm2). The number of blood vessels per 1 mm2 was counted and compared among men and women, femur or tibia, with and without free flap surgery, antibiotic impregnation to the cement, osteogenesis inside the membrane, smoking, and diabetes mellitus. The number of blood vessels within the same patient was compared among different time points. Correlation analysis was performed among blood vessel numbers and patient age, duration of cement spacer placement, and histological grading scales (inflammation, foreign body reaction, and fibrosis). RESULTS: IM formation with rich vascularity and some inflammation, foreign body reaction, and fibrosis were histologically confirmed in all patients. We found 37.4 ± 19.1 blood vessels per 1 mm2. The number of blood vessels was significantly lower in patients with than in those without free flap surgery; it was higher in patients with osteogenesis inside the IM. No significant correlations were found in any of the analyses. CONCLUSION: Sex, patient age, smoking, diabetes mellitus, femur or tibia, duration of cement spacer placement, and antibiotic impregnation to the cement did not affect IM vascularization. IM vascularization was reduced in patients with than in those without free flap surgery.

The Dual Role of Vitamin K2 in "Bone-Vascular Crosstalk": Opposite Effects on Bone Loss and Vascular Calcification.

Osteoporosis (OP) and vascular calcification (VC) represent relevant health problems that frequently coexist in the elderly population. Traditionally, they have been considered independent processes, and mainly age-related. However, an increasing number of studies have reported their possible direct correlation, commonly defined as "bone-vascular crosstalk". Vitamin K2 (VitK2), a family of several natural isoforms also known as menaquinones (MK), has recently received particular attention for its role in maintaining calcium homeostasis. In particular, VitK2 deficiency seems to be responsible of the so-called "calcium paradox" phenomenon, characterized by low calcium deposition in the bone and its accumulation in the vessel wall. Since these events may have important clinical consequences, and the role of VitK2 in bone-vascular crosstalk has only partially been explained, this review focuses on its effects on the bone and vascular system by providing a more recent literature update. Overall, the findings reported here propose the VitK2 family as natural bioactive molecules that could be able to play an important role in the prevention of bone loss and vascular calcification, thus encouraging further in-depth studies to achieve its use as a dietary food supplement.

Bone Fragment Co-transplantation Alongside Bone Marrow Aspirate Infusion Protects Kidney Transplant Recipients.

Integration of non-vascularized bone grafting and bone marrow aspirate infusion in transplantation may provide clinical benefit. Here we have incorporated bone fragment co-transplantation and bone marrow aspirate infusion (BF-BM) into living kidney transplantation (LKT). Twenty LKT recipients receiving bone fragments and bone marrow aspirates donated from their corresponding donors were enrolled into a retrospective study. A contemporaneous control group was formed of 38 out of 128 conventional LKT recipients, selected using propensity score matching by a 1:2 Greedy algorithm. Ultrasonography, contrast-enhanced ultrasonography (US/CEUS) and SPECT/CT showed that the co-transplanted bone fragments remained viable for 6 months, subsequently shrank, and finally degenerated 10 months post-transplantation. BF-BM resulted in earlier kidney recovery and more robust long-term kidney function. Throughout 5 years of follow-up, BF-BM had regulatory effects on dendritic cells (DCs), T helper (Th1/Th2) cells and regulatory T cells (Tregs). Both alloantigen-specific lymphocyte proliferation and panel reactive antibody levels were negative in all recipients with or without BF-BM. In addition, the BF-BM group experienced few complications during the 5-year follow-up (as did the donors)-this was not different from the controls. In conclusion, BF-BM is safe and benefits recipients by protecting the kidney and regulating the immune response.

Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice.

Rationale: Mechanisms underlying the compromised bone formation in type 1 diabetes mellitus (T1DM), which causes bone fragility and frequent fractures, remain poorly understood. Recent advances in organ-specific vascular endothelial cells (ECs) identify type H blood vessel injury in the bone, which actively direct osteogenesis, as a possible player. Methods: T1DM was induced in mice by streptozotocin (STZ) injection in two severity degrees. Bony endothelium, the coupling of angiogenesis and osteogenesis, and bone mass quality were evaluated. Insulin, antioxidants, and NADPH oxidase (NOX) inhibitors were administered to diabetic animals to investigate possible mechanisms and design therapeutic strategies. Results: T1DM in mice led to the holistic abnormality of the vascular system in the bone, especially type H vessels, resulting in the uncoupling of angiogenesis and osteogenesis and inhibition of bone formation. The severity of osteopathy was positively related to glycemic levels. These pathological changes were attenuated by early-started, but not late-started, insulin therapy. ECs in diabetic bones showed significantly higher levels of reactive oxygen species (ROS) and NOX 1 and 2. Impairments of bone vessels and bone mass were effectively ameliorated by treatment with anti-oxidants or NOX2 inhibitors, but not by a NOX1/4 inhibitor. GSK2795039 (GSK), a NOX2 inhibitor, significantly supplemented the insulin effect on the diabetic bone. Conclusions: Diabetic osteopathy could be a chronic microvascular complication of T1DM. The impairment of type H vessels by NOX2-mediated endothelial oxidative stress might be an important contributor that can serve as a therapeutic target for T1DM-induced osteopathy.

Progenitor cell-derived exosomes endowed with VEGF plasmids enhance osteogenic induction and vascular remodeling in large segmental bone defects.

Large segmental bone regeneration remains a great challenge due to the lack of vascularization in newly formed bone. Conventional strategies primarily combine bone scaffolds with seed cells and growth factors to modulate osteogenesis and angiogenesis. Nevertheless, cell-based therapies have some intrinsic issues regarding immunogenicity, tumorigenesis, bioactivity and off-the-shelf transplantation. Exosomes are nano-sized (50-200 nm) extracellular vesicles with a complex composition of proteins, nucleic acids and lipids, which are attractive as therapeutic nanoparticles for disease treatment. Exosomes also have huge potential as desirable drug/gene delivery vectors in the field of regenerative medicine due to their excellent biocompatibility and efficient cellular internalization. Methods: We developed a cell-free tissue engineering system using functional exosomes in place of seed cells. Gene-activated engineered exosomes were constructed by using ATDC5-derived exosomes to encapsulate the VEGF gene. The specific exosomal anchor peptide CP05 acted as a flexible linker and effectively combined the engineered exosome nanoparticles with 3D-printed porous bone scaffolds. Results: Our findings demonstrated that engineered exosomes play dual roles as an osteogenic matrix to induce the osteogenic differentiation of mesenchymal stem cells and as a gene vector to controllably release the VEGF gene to remodel the vascular system. In vivo evaluation further verified that the engineered exosome-mediated bone scaffolds could effectively induce the bulk of vascularized bone regeneration. Conclusion: In our current work, we designed specifically engineered exosomes based on the requirements of vascularized bone repair in segmental bone defects. This work simultaneously illuminates the potential of functional exosomes in acellular tissue engineering.