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Exosomes secreted from human-derived adipose stem cells prevent progression of osteonecrosis of the femoral head

Journal of Orthopaedic Surgery and Research volume 19, Article number: 793 (2024) Cite this article

A Correction to this article was published on 24 April 2025

This article has been updated

Abstract

Background

Osteonecrosis of the femoral head (ONFH) primarily affects young individuals and is a leading cause of total hip arthroplasty in this population. Joint-preserving regenerative therapies involving core decompression (CD), enhanced with cells, growth factors, and bone substitutes, have been developed but lack extensive validation. Exosomes are emerging as a promising regenerative therapy. Human adipose stem cell (hADSC)-derived exosomes exhibit angiogenic and wound-healing effects on damaged and diseased tissues, suggesting their potential efficacy in treating early-stage ONFH. We aimed to investigate the efficacy of hADSC-derived exosomes based on CD in a medium-sized animal model (rabbit).

Methods

Exosomes were extracted using the ultrafiltration filter technique from the culture supernatants of two types of hADSCs. Characterization of exosomes was performed through nanoparticle tracking analysis, transmission electron microscopy, and the detection of specific biomarkers (CD9, CD63, and CD81) by western blotting. Eighteen rabbits underwent surgical vascular occlusion and intramuscular corticosteroid injections to induce ONFH. Concurrently, CD treatment with local administration of hADSC-derived exosomes (exosome group) or saline (control group) was performed. Femoral heads were harvested at 4, 8, and 12 weeks postoperatively and evaluated using micro-computed tomography and tissue staining to assess the protective effects on osteonecrosis, angiogenesis, and osteogenesis.

Results

Exosomes had average particle concentrations of 1.8 × 1012 or 1.8 × 109 particles/mL, with particle size distributions averaging 61.2 ± 14.7 or 123.1 ± 46.3 nm, and were confirmed by specific biomarkers. The exosome group exhibited a significant reduction in the severe progression of ONFH to stages 3 or 4 of the modified Ficat and Arlet classification, compared to the control group, which had four cases of stages 3 or 4. The exosome group showed significantly fewer empty lacunae in the subchondral bone area (p < 0.05) and significantly less articular cartilage injury (p < 0.05) compared to the corresponding in the control group. There were no significant differences in the microvessel number, bone trabecular structure, or volume of new bone in the medial region of the CD.

Conclusions

hADSC-derived exosomes can prevent the progression of ONFH by inhibiting osteonecrosis and cartilage damage. The ultrafiltration filter technique is effective for exosome extraction, indicating that exosomes hold potential as a therapeutic agent for ONFH.

Background

Osteonecrosis of the femoral head (ONFH) is a rare joint disease characterized by hip pain, femoral head collapse, and gait disturbance, stemming from the death of bone tissue. Although the pathogenesis of ONFH is marked by the presence of empty lacunae in osteocytes, its etiology remains incompletely understood. Systemic corticosteroid therapy is a major risk factor for ONFH, involving oxidative stress, vascular endothelial damage, fat embolism, apoptosis, decreased osteoblast production, and increased osteoclast activity [1, 2].

In a previous study involving 505 ONFH hips, it was found that 85% of hips with large extensive types developed radiological collapse within 5 years. Moreover, half of the ONFH hips had already collapsed at the initial diagnosis, and 63% of the cases were bilateral [3]. Another study reported that 59% of untreated ONFH cases progressed to symptoms or collapse [4]. Most ONFH cases eventually progress to secondary osteoarthritis (OA), often necessitating total hip arthroplasty (THA) [5]. Although THA is a highly successful orthopedic surgery, it is not ideal for young patients with ONFH due to restrictions on sports activities, high dislocation risk, lifelong periprosthetic joint infection risk, potential for revision surgeries, and high medical costs. Thus, the primary goal of ONFH treatment is to preserve the hip joint, prevent femoral head collapse, and delay the need for THA. Historically, core decompression (CD) has been a major joint-preserving surgery in the West.

CD is favored due to its minimally invasive nature, ease of percutaneous execution, and requirement for a short hospitalization period. Since around 2000, various regenerative therapies based on CD have emerged for treating ONFH. Recently, the biological augmentation of CD with cells or growth factors has been explored as a next-generation treatment for ONFH [6].

The use of autologous bone marrow or bone marrow mesenchymal stem cells (BMMSCs) has shown positive results in the treatment of ONFH, as demonstrated in both animal studies and clinical trials [7,8,9]. Adipose-derived stem cells (ADSCs) can enhance cell growth and differentiation, angiogenesis, and osteogenesis through secreted factors with paracrine mechanisms [10,11,12]. Recent studies have focused on the function of exosomes as intercellular messengers, serving as an alternative to stem cells [13]. Exosomes are nano-sized extracellular vesicles ranging from 50 nm to 200 nm [14, 15]. They encapsulate messenger RNA (mRNA) and microRNAs that perform various roles in gene regulation, immune regulation, and tissue repair [16]. Animal studies have shown that exosomes derived from mesenchymal stem cells (MSCs) are effective in treating ONFH [17, 18]. However, the effect of human ADSC-derived exosomes (hADSC-Exos) on ONFH in medium-sized animals remains uncertain. In this study, as a preclinical model for potential future clinical application, we aimed to confirm the inhibitory effect of hADSC-Exos on the progression of ONFH in a medium-sized animal model.

Methods

Exosome purification

Collection of culture supernatants

In this study, exosomes were isolated from the culture supernatants of two kinds of hADSCs to confirm the effectiveness of the ultrafiltration filter technique in exosome extraction. One type of exosome (Exosome X) was derived from hADSCs of human abdominal subcutaneous fat. ADSCs were collected from a patient with knee OA who had provided written informed consent. hADSCs were isolated from the fat tissue using a non-woven fabric and cultured in an optimized medium with 1–4% autologous serum for 3–4 weeks at 37 °C with 5% CO2 to a maximum of approximately 1 × 108 cells. Then, the culture medium was changed to Nutri Stem media with 2–4% autologous serum, and the supernatant was collected after centrifugation.

The other type of exosome (Exosome Y) was derived from Poietics™ hADSC (Lonza, Basel, Switzerland). After the medium was removed from the subconfluent cell culture, hADSCs were washed once with a cell wash solution and cultured in ADSCs Growth Medium BulletKit™ (Lonza) until they reached 40–50% confluency. When hADSCs increased to 40–50% confluency, the culture medium was changed to adipocyte culture medium (phenol red, serum-free) (ZenBio, Durham, NC, USA) and the cells were cultured for 4 days. Following centrifugation at 1,000 rpm for 5 min, the supernatant was collected and filtered through a Rapid-Flow Filter (Thermo Fisher Scientific, Waltmam, MA, USA) to remove whole cells and cell debris.

Exosome isolation and purification

Exosomes were isolated by the ultrafiltration filter technique from the cell culture supernatant of two types of ADSCs. The cell culture supernatants were concentrated directly without solvent substitution. Initially, the culture supernatant was collected using a 0.22-µm pre-filter. The collected solution was concentrated 10-fold using an ultrafiltration filter. Finally, the concentrated solution was passed through a 0.22-µm final filter.

Identification of exosomes

Nanoparticle tracking analysis (NTA)

Exosomes were analyzed for distribution and concentration using NTA with the nano sight NS300 instrument (Malvern Panalytical, Malvern, UK).

Western blotting

Western blotting was used to confirm the presence of exosome protein markers of CD9, CD63, and CD81. Specifically, 5 mL of each sample was centrifuged at 100,000 x g for 70 min at 4 °C. The supernatant was removed, and each sample was centrifuged again with 5 mL of phosphate-buffered saline (PBS) (100,000 x g, 70 min, 4 °C). The supernatant was removed and 100 µL of radio-immunoprecipitation assay buffer containing protease inhibitor was added. The samples were incubated under ice cold for 30 min, centrifuged at 15,500 × g for 10 min at 4 °C, and the supernatant was collected as lysate.

Subsequently, 20 µL of lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 20 mA for 80 min and transferred to polyvinylidene difluoride membrane (Cytiva, Tokyo, Japan) at 120 mA for 60 min. The membrane was blocked with 20% tris-buffered saline tween for 60 min at room temperature. The membranes were incubated with anti-CD9, CD63, and CD81 antibodies (1:500 or 1:1000) (Cosmo Bio, Tokyo, Japan) for 1 h at room temperature. Then, the membranes were incubated with HRP-conjugated anti-Mouse secondary antibody (1:40000) (Thermo Fisher Scientific) for 60 min at room temperature, visualized with ImmunoStar® Zeta (Wako, Osaka, Japan), and the results were analyzed with X-ray film (Hyperfilm ECL; Cytiva).

Transmission electron microscopy (TEM)

The morphology of hADSCs-Exos was observed using TEM with the negative staining method. A collodion membrane (Formvar) membrane-attached grid (200 mesh Cu Nisshin-em, Tokyo, Japan) was hydrophilized. The grid was placed on top of a 5-µL droplet of exosome solution pipetted onto a parafilm for 10 min. Then, exosome solution on the grid was absorbed using filter paper. Next, the grid was placed on 5 µL of 1% uranium solution on a parafilm for 1 min for negative staining. After drying, the morphology of the hADSC-Exos was observed using TEM (JEOL, Tokyo, Japan).

Animal experiments

Preparation of animals and protocol

The experiments were approved by the Ethics Animal Research Committee at Graduate School of Medicine, Kyoto University, Japan. All procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. Eighteen adult female Japanese white rabbits aged ≥ 25 weeks (weight, 2.6–3.7 kg) were used in this study. Each rabbit had its two hind legs designated as the exosome treatment group and the control group, respectively.

Following the induction of ischemia and administration of corticosteroids, hADSC-Exos or saline were locally administered to femoral heads. Nine rabbits were treated with exosome from each type of hADSCs.

At 4, 8, and 12 weeks postoperatively, six rabbits (exosome group; exosome X: n = 3 and exosome Y: n = 3 at each endpoint) were randomly chosen and sacrificed. The femurs were harvested for subsequent analysis of micro-computed tomography (µCT) and histological evaluation (Fig. 1). An additional healthy sacrificed sample was included for comparison with the treatment group based on imaging.

Fig. 1
figure 1

Flowchart of this study. Eighteen adult female rabbits were used in this study. The OFNH model of rabbits was created with corticosteroid injection and ischemic operation. Each rabbit had one hind leg assigned to the exosome group and the other hind leg to the control group. Two kinds of hADSC-Exos were administered to nine rabbits per group. At 4, 8, and 12 weeks, six rabbits were randomly chosen (three rabbits for each exosome) for sacrifice and assessment. hADSC-Exos, human adipose stem cell derived exosome

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Surgical procedure

Rabbits were anesthetized with an intravenous dose of 15 mg/kg of thiopental and further anesthetized via isoflurane inhalation. Local anesthesia was achieved using a 1% lidocaine solution. The rabbit ONFH model was created following a previously reported protocol [19], involving the administration of high-dose corticosteroids (40 mg/kg methylprednisolone) and an ischemic procedure with electrocoagulation of the femoral neck on the same day.

In all cases, a 3-cm skin incision was made along the greater trochanter to access the posterolateral side using a posterolateral technique. The ischemic procedure involved electrocoagulation to disrupt the blood flow to the femoral head by targeting the rotator arteries at the femoral neck. Subsequently, a total dose of 40 mg/kg methylprednisolone was divided equally and injected into the gluteal muscles on both sides (20 mg/kg per side).

CD with the administration of hADSC-Exos or 0.9% saline was performed on the same day as the ONFH procedure. A 2-mm diameter tunnel parallel to the femoral neck at the femoral head was created using a 2-mm diameter dental bur, ensuring not to exceed the boundary surface of the femoral head cartilage.

Especially, 33 mg of gelatin hydrogel (GelArt, Shibuya Industry, Ishikawa, Japan) was mixed with 200 µL of hADSC-Exos diluted three times with 0.9% saline (dosage; exosome X: 1.19 × 1011 particles, exosome Y: 1.22 × 108 particles) for the exosome group, and with 200 µL of 0.9% saline for the control group. The difference in particle concentrations between exosome X and Y reflects the maximum concentration that could be achieved using the ultrafiltration filter technique for each exosome type. This gelatin hydrogel consisted of gelatin particles derived from alkali-treated porcine skin gelatin by particularizing and cross-linking. To facilitate administration of the gelatin hydrogel into the femoral head, a small amount of saline was added to ensure proper hydration and delivery. The exosome with gelatin hydrogel was administered topically to the femoral head using an 18G needle through the bone tunnel. The incision was sutured in one layer. The rabbits were housed in cages and allowed to move freely post-procedure.

Methods of analysis of radiographic and histology

X-ray and µCT analysis

The harvested femoral heads underwent X-ray and µCT scanning to evaluate trabecular bone structure, cartilage status, and bone formation within the CD area. Scans were conducted using a µCT system (Skyscan 1275, Bruker, Billerica, MA, USA) with a voxel resolution of 30 μm, 75 µA, 65 kV, and an angular step of 0.2° averaged over two frames.

The modified Ficat and Arlet classification was utilized to stage ONFH based on anteroposterior and lateral radiographic appearances of the femoral head [20]. This widely accepted classification system categorizes ONFH into stages: stage I (normal), stage II (cystic lesion and sclerotic change), stage III (crescent sign or subchondral fracture), and stage IV (complete collapse and secondary OA).

For evaluation of subchondral structure, bone trabecular structure was measured within a 2-mm quadrangle area located 0.3–0.5 mm below the femoral head’s surface along the femoral axis [21]. The bone volume of the new bone within the CD borehole area was also assessed [19]. A slice showing optimal visibility of the borehole was selected, and threshold values were determined for each sample using the Hounsfield Unit value that maximized class variance between water and bone readings in the µCT scans, facilitating differentiation between bone and marrow.

Histological and immunohistochemical analysis

The upper portion of the collected femurs was fixed in 4% paraformaldehyde for 1 week. Subsequently, the samples were demineralized in 0.5 M ethylenediaminetetraacetic acid (pH 7.4), followed by paraffin embedding and sectioning into 0.5-µm thick slices aligned with the femoral shaft.

Sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E), as well as safranin O. Digital microscopy (BZ-X710, Keyence, Osaka, Japan) was employed for analysis. Bone regeneration was evaluated by calculating the ratio of empty lacunae to normal bone cells. This assessment involved analyzing an average of four 20x magnification views from the subchondral bone and three 20x magnification views from the tunnel area [22]. The degree of articular cartilage degeneration was determined using a semiquantitative modified Mankin score [23].

Immunostaining for CD31 (1:400, Abcam, Cambridge, UK) was performed to quantify blood vessels in the subchondral bone. Briefly, sections were rehydrated, subjected to antigen retrieval in Hist VT One (pH 7.0) at 65 °C for 20 min, and blocked with 5% goat serum in PBS (pH 7.4) for 60 min at room temperature. The sections were then incubated overnight at 4 °C with primary antibodies, followed by a 1-h incubation with secondary antibody (1:400, Abcam). DAPI staining was used to label nuclei, and sections were examined using digital microscopy. Vessels were identified by positive CD31 staining and characteristic structural features [24]. Seven fields were photographed at 20x magnification for each staining method, and vessel counts were determined for each field.

Statistical analysis

All data were analyzed using JMP Pro 15.2 (SAS Institute, Cary, NC, USA). The level of significance was set at p < 0.05. Wilcoxon signed-rank test and chi-square test were used. Data are presented as means ± standard deviations.

Results

ADSC-Exos characterization and content identification

NanoSight analysis revealed that these particles had a diameter distribution with an average dimension of 61.2 ± 14.7 nm for exosome X and 123.1 ± 46.3 nm for exosome Y (Fig. 2a), with a mean concentration of 1.8 × 1012 particles/mL for exosome X and 1.8 × 109 particles/mL for exosome Y. TEM showed spherical extracellular vesicles measuring approximately 40 nm in diameter for exosome X and 100 nm for exosome Y (Fig. 2b). Western blotting showed the presence of the typical exosome biomarkers, including CD9, CD63, and CD81, on these particles (Fig. 2c). Collectively, these results verify the identity of these particles as exosomes.

Fig. 2
figure 2

Characterization of exosomes. (a) Particle size distributions and concentration of exosome determined by nanoparticle tracking analysis. (b) Transmission Electron Microscopy with negative staining. Scale bar; 100 nm. (c) Western blotting of the positive specific biomarkers of CD9, CD63, and CD81. X, exosome X; Y, exosome Y

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Staging of ONFH and assessment of radiographic

Figure 3 presents representative X-ray and µCT images at 8 weeks. In the exosome group, X-ray and macroscopic examinations indicated that the femoral head maintained its shape with a reddish surface. In contrast, the control group exhibited a white to yellowish femoral head with subchondral cysts and degenerated cartilage. The results of X-ray and µCT analyses are summarized in Table 1. According to the modified Ficat and Arlet classification, the control group consisted of stage 1 (n = 12), stage 2 (n = 2), stage3 (n = 1), and stage 4 (n = 3), whereas the exosome group showed stage 1 (n = 15), and stage 2 (n = 3) at all evaluated time points. Progression of ONFH was limited to stage 2 or lower in the exosome group, whereas three cases progressed to stage 4 in the control group (Fig. 3). Although not statistically significant, the grade tended to be lower in the exosome group at each time point. Moreover, µCT evaluation of trabecular structure in the subchondral bone and bone formation revealed no significant differences between the exosome group and the control group at 12 weeks. Trabecular structure did not show discernible changes over the weeks, and bone formation was comparable between the groups (Supplementary Fig. 1).

Fig. 3
figure 3

Staging of ONFH and assessment of radiographic. Representative appearance and µCT images at 8 weeks are shown. In exosome group, the shape of the femoral head maintained its shape with a reddish surface. According to the modified Ficat and Arlet classification, the stage was 0, and osteogenesis was observed in the tunnel (white circle) on µCT. In contrast, the control group exhibited complete fracture of the femoral head and articular destruction, with a modified Ficat and Arlet classification of stage 4. µCT, micro-computed tomography

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Table 1 Assessment of X-ray and µCT
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Histological and immunohistochemical analysis

Figure 4a shows the results of H&E staining of the subchondral bone. In the control group, thrombosis was observed at 4 weeks, followed by bone marrow fibrosis at 8 weeks, and numerous empty lacunae were evident at 12 weeks. At 4 weeks, there were fewer empty lacunae in both groups, significantly fewer in the exosome group (p < 0.01) (Supplementary Fig. 2). The exosome group demonstrated suppression of the increase in empty lacunae over time. By 12 weeks, the exosome group exhibited 17.4% empty lacunae compared to 46.2% in the control group in the subchondral area. Significant differences were observed between the groups, with fewer empty lacunae present in both the subchondral area and the area surrounding the tunnel in the exosome group (Table 2).

Fig. 4
figure 4

Histological assessment. (a) Representative images of H&E staining at the subchondral area. In the control group, the bone marrow becomes necrotic and empty lacunae were observed as the weeks progressed. Additionally, there was subchondral fracture in the control group (blue arrow). In the exosome group, normal bone marrow was present, and no empty lacunae were observed. The triangle shows empty lacunae, and the black arrow shows thrombosis, the orange arrow indicates bone tunnel. (b) Safranin O staining of the articular cartilage. In the exosome group, bone cartilage and morphology were preserved, although staining was reduced. (c) Representative images of CD31 immunohistochemistry. The yellow arrows show vessels with CD31 positive. CD31-positive cells are observed in the exosome group. Scale bar: 500 μm (yellow),100 μm (black). H&E, hematoxylin and eosin; VM, visible marrow; NM, necrotic marrow

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Table 2 Evaluation of empty lacunae and angiogenesis
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Figure 4b shows representative images of safranin O staining at the femoral articular cartilage. The results showed a decrease of chondrocytes and progression of cartilage degeneration. The modified Mankin scores were predominantly lower in the exosome group compared to those in the control group at 4 weeks (Supplementary Fig. 2), and this difference persisted, with significantly lower scores in the exosome group at 12 weeks (p = 0.048) (Table 2).

Figure 4c shows representative images of CD31 immunostaining in the subchondral area. In the control group, the bone marrow was fibrotic, and there were less vessels at 12 weeks. Conversely, the exosome group maintained normal bone marrow and preserved normal vessel structure. Quantitative analysis of CD31-positive vessels indicated a decrease in vessel numbers from 4 weeks onward in the control group in the subchondral area (Supplementary Fig. 2). However, the number of vessels tended to be maintained in the subchondral area in the exosome group even if time passes. There was no significant difference in the number of vessels at 12 weeks (Table 2).

Discussion

The treatment of ONFH remains a formidable challenge for orthopedic surgeons, prompting ongoing efforts to identify optimal therapies. In the context of preventing corticosteroid-associated ONFH, certain agents, such as anticoagulants, lipid-lowering agents, and antioxidants, have demonstrated preventive effects in animal studies. However, clinical trials are lacking, representing a significant hurdle in this area of research [25].

Reports have indicated that ONFH can manifest within 1 weeks following corticosteroid administration, highlighting the critical window for preventive measures immediately preceding corticosteroid use [26, 27]. In our study, we administered exosomes concurrently with the induction of the ONFH model to evaluate their potential preventive effects.

ADSC-Exos are emerging as promising candidates due to their ability to facilitate angiogenesis and osteogenesis via paracrine mechanisms. ADSCs, sourced from adipose tissue, possess attributes, such as high proliferative capacity, widespread availability, and multipotency [28, 29]. However, direct application of ADSCs is hampered by challenges, including lengthy cell culture requirements, concerns over ectopic differentiation, and potential immune responses [30].

Exosomes derived from ADSCs offer a viable alternative therapy by harnessing their paracrine-mediated effects. They demonstrate therapeutic efficacy comparable to ADSCs without the associated risks [11]. Moreover, exosome therapy presents several advantages over cell-based treatments. Exosomes exhibit lower immunogenicity and toxicity, making them safer and more feasible for long-term use [31]. They also offer easier storage and greater cost-effectiveness compared to cell-based therapies [32].

The main methods of exosome extraction include ultracentrifugation, size-based techniques, and bead-based immunoprecipitation [33]. Ultrafiltration is a method for purifying exosomes from culture supernatants using only filtration techniques, without ultracentrifugation or special agents. This process takes less time than ultracentrifugation and does not require special equipment, potentially increasing work efficiency and yielding a high recovery rate of exosomes [34, 35]. In this study, we confirmed that highly concentration of exosomes can be extracted from the culture supernatants of two types of hADSC by filter ultrafiltration. Therefore, ultrafiltration is a useful method in exosome retrieval.

In this study, hADSC-Exos demonstrated early reduction of empty lacunae and prevented the progression of ONFH. Moghassemi et al. reported that corticosteroid-induced osteonecrosis is primarily attributed to thrombus-associated ischemia caused by femoral head congestion and induction of apoptosis due to oxidative stress [36]. In vitro studies have demonstrated that ADSC-Exos mitigate osteocyte apoptosis and prevent osteoclast activity by decreasing the expression of receptor activator of nuclear factor kappa b ligand [37]. Additionally, extracellular vesicles from mesenchymal stem cell-conditioned medium act as senomorphics, downregulating senescence-related genes, such as p16INK4a, p21, and p53 [38]. Therefore, hADSC-Exos likely inhibit ONFH progression through protective and anti-apoptotic effects on osteocytes immediately upon administration.

In this study, the exosome-treated group tended to exhibit lower stages in the Modified Ficat and Arlet classification. However, the small sample size (n = 6) used for evaluating empty lacunae and bone mass may limit the ability to detect significant differences in disease stage. Future studies with larger sample sizes could provide more conclusive evidence regarding the efficacy of exosomes in reducing disease severity.

Angiogenesis is critical for prevention of osteonecrosis and bone regeneration. Increased angiogenesis has shown a therapeutic effect of ONFH in steroid-induced ONFH models [39, 40]. ADSC-Exos reportedly promotes angiogenesis in many tissues by enhancing secretion of vascular endothelial growth factor from vascular endothelial cells [41, 42]. Exosomes reportedly inhibit thrombus formation by promoting plasminogen activator inhibitor 1 expression via microRNAs [43]. These effects of thrombus inhibition, and promotion of angiogenesis in ischemic areas may prevent progression of ONFH. Therefore, we expected that angiogenesis would be promoted in the exosome group. Vascular reduction was suppressed in the exosome group, but there was variability in the amount of blood vessels and no significant promotion was observed in this study. This may be attributed to the limited angiogenic effect of exosomes in hypoxic conditions.

In this study, inhibition of secondary OA changes by hADSC-Exos was observed. ONFH is often progressive and frequently culminates in secondary OA. Steroid-induced factors have been shown to play a significant role in this process. Lin et al. reported that exposure to methylprednisolone leads to a decrease in the expression of miR-30b-5p in chondrocytes, which subsequently causes an increase in runt-related transcription factor 2 levels {Lin, 2022 #724}. Runx2 promotes chondrocyte hypertrophy and apoptosis, accelerating cartilage degradation. Additionally, Hofstaetter et al. suggested that the resorption of necrotic trabeculae weakens the structural support of the femoral head, contributing to the collapse of ONFH and potentially leading to secondary OA {Hofstaetter, 2009 #727}. Recent reports have suggested that hADSC-Exos can downregulate inflammation and oxidative stress, and protect chondrocytes from apoptosis [44,45,46]. This may influence the chondrocyte metabolism associated with joint destruction and inhibit the progression of cartilage destruction.

In the early stage of ONFH, the trabecular thickness tends to decrease, but no significant change of the cancellous structure was observed using µCT evaluation within 42 weeks after glucocorticoid administration [2]. The relatively short 12-week evaluation period in this study may have been insufficient to detect these expected structural changes. Additionally, the localized effects of CD may have influenced the accuracy of trabecular bone measurements. Although the images were captured at a voxel resolution of 30 μm, a higher-resolution voxel size tailored for subchondral bone microstructure could have provided a more precise assessment. Furthermore, µCT assesses bone density based on signal intensity, and factors such as cellular infiltration, appearing as areas of increased density, might have contributed to the lack of significant differences in trabecular structure and bone formation.

Wang et al. reported that the administration of exosomes alone show no significant effect likely due to their rapid excretion [47]. Addressing this challenge, researchers have explored methods to establish sustained delivery systems for exosomes [48]. For instance, it has been demonstrated that incorporating FGF-2 into gelatin hydrogel enables long-term sustained release of FGF-2, thereby contributing to the improvement of ONFH [49, 50]. Several studies have reported that encapsulating exosomes in hydrogels can maintain local concentration for long periods of time, thus providing therapeutic benefits [47, 51, 52]. In this study, exosomes were encapsulated in a gelatin gel and placed locally. However, the release kinetics of exosomes from the gelatin hydrogel were not investigated. Further research is needed to assess the efficacy of gel-based delivery systems for exosomes in treating ONFH.

This study had several limitations. First, the mechanism behind the effects of exosomes were not examined. In recent studies, the effects and mechanisms of various microRNAs in exosomes were studied. Optimization of miRNAs in the exosome membrane may be a clue to exosome-based therapies. Second, two types of hADSC-Exos were used, and the dosage of each exosome was different. There is a lack of discussion regarding the optimal dosage and delivery system of exosomes, which remains a topic for future research. Third, the small sample size at each endpoint in our study may have limited our ability to detect significant differences in the parameters examined. Fourth, we only evaluated short-term outcomes up to 12 weeks postoperatively. Future studies should include longer follow-up periods to assess the long-term efficacy and durability of the interventions.

Conclusions

In conclusion, hADSC-Exos decreased the empty lacunae and cartilage damage and prevented the progression of ONFH. Our findings suggest that the application of ADSC-Exo may be a promising therapeutic tool in the treatment of ONFH.

Data availability

No datasets were generated or analysed during the current study.

Change history

Abbreviations

ADSCs:

Adipose-derived stem cells

BMMSCs:

Bone marrow mesenchymal stem cells

CD:

Core decompression

hADSC:

Exos-Human ADSC-derived exosomes

NTA:

Nanoparticle tracking analysis

mRNA:

Messenger RNA

MSCs:

Mesenchymal stem cells

OA:

Osteoarthritis

ONFH:

Osteonecrosis of the femoral head

TEM:

Transmission electron microscopy

THA:

Total hip arthroplasty

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Acknowledgements

The electron microscopy study was supported by Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University.

Funding

This work was supported in part by grants from Kyoto University Open Innovation Institute and Kyoto City Innovation Creation Support Center (to K.Y.).

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, Sakyo, Kyoto, 606- 8507, Japan

    Tatsuhito Ikezaki, Yutaka Kuroda, Toshiyuki Kawai, Yaichiro Okuzu, Yugo Morita & Shuichi Matsuda

  2. Department of Orthopaedic Surgery, Kindai University Graduate School of Medicine, Onohigashi, Osakasayama, Osaka, 589-8511, Japan

    Koji Goto

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  1. Tatsuhito Ikezaki
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Contributions

All authors contributed to the study conception and design. Animal operation was done by TI and YK. Data collection was done by TI. Material preparation and analysis were performed by TI and YK. The first draft of the manuscript was written by TI, YK. All authors commented on previous versions of the manuscript. The final manuscript was written by TI and YK. All authors read and approved the final manuscript.

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Correspondence to Tatsuhito Ikezaki.

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The experiments were approved by the Ethics Animal Research Committee at Graduate School of Medicine, Kyoto University, Japan. All procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals.

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The authors declare no competing interests.

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Ikezaki, T., Kuroda, Y., Kawai, T. et al. Exosomes secreted from human-derived adipose stem cells prevent progression of osteonecrosis of the femoral head. J Orthop Surg Res 19, 793 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13018-024-05267-w

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Keywords

Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X