Abstract

   The early treatment of Osteonecrosis of Femoral Head (ONFH) remains a
   clinical challenge. Conventional Bone Marrow Mesenchymal Stem Cell
   (BMSC) injection methods often result in unsatisfactory outcomes due to
   mechanical cell damage, low cell survival and retention rates,
   inadequate cell matrix accumulation, and poor intercellular
   interaction. In this study, we employed a novel cell carrier material
   termed "3D Microscaffold" to deliver BMSCs, addressing these issues and
   enhancing the therapeutic effects of cell therapy for ONFH. We injected
   3D microscaffold loaded with low-dose BMSCs or free high-dose BMSCs
   into the femoral heads of ONFH rats and assessed therapeutic effects
   using imaging, serology, histology, and immunohistochemistry. To
   understand the mechanism of efficacy, we established a co-culture model
   of human osteoblasts and BMSCs, followed by cell proliferation and
   activity detection, flow cytometry analysis, Quantitative RT-PCR, and
   Western blotting. Additionally, RNA sequencing was performed on femoral
   head tissues. Results showed that the 3D microscaffold with low-dose
   BMSCs had a therapeutic effect comparable to high-dose free BMSCs.
   Osteoblasts in the 3D microscaffold group exhibited superior phenotypes
   compared to the non-3D microscaffold group. Furthermore, we have, for
   the first time, preliminarily validated that the low-dose BMSCs-loaded
   3D microscaffolds may promote the repair of femoral head necrosis
   through the synergistic action of the MAPK and Hippo signaling
   pathways.

   Keywords: Osteonecrosis of the femoral head, Stem cell, Microscaffold

Graphical abstract

   [47]Image 1
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1. Introduction

   ONFH (osteonecrosis of the femoral head) represents a prevalent and
   challenging orthopedic disorder. In its advanced stages, ONFH
   frequently precipitates femoral head collapse, concomitant with the
   onset of secondary osteoarthritis [[49]1,[50]2]. The pathogenesis of
   osteonecrosis of the ONFH remains a topic of ongoing debate and
   controversy. In addition to traumatic factors, common etiological
   factors encompass the misuse of glucocorticoids, alcohol abuse,
   coagulation dysfunction, sickle cell anemia, other hemoglobinopathies,
   inflammation, and autoimmune disorders. Among these, steroid-induced
   femoral head osteonecrosis prevails as the most frequently observed
   condition [[51]3]. The current diagnosis and treatment of ONFH
   primarily rely on the ARCO staging system and individual patient
   factors. For patients in stage I and II according to the ARCO
   classification, conservation treatments such as immobilization,
   appropriate traction, medication, core decompression, osteotomy, and
   vascularized or non-vascularized bone grafting are commonly employed.
   The primary objectives are to alleviate pain and slow down the
   progression of necrosis. On the other hand, for patients in stage III
   and IV, total hip arthroplasty (THA) is often required. THA involves
   the complete replacement of the hip joint [[52][4], [53][5], [54][6]].
   However, none of these methods have demonstrated the ability to reverse
   and cure early-stage ONFH. Furthermore, each method has its own
   limitations. For instance, while immobilization and traction, as well
   as medication, carry a low risk of harm to patients, there is still
   debate regarding their effectiveness in alleviating long-term pain and
   slowing disease progression [[55]7]. Various hip preservation surgeries
   may require patients to remain bedridden for an extended period after
   the procedure. Additionally, the low success rates and significant harm
   to patients associated with these surgeries have remained persistent
   concerns for many clinicians in the field. THA is the ultimate
   treatment option for patients with ONFH [[56]8]. However, the lifespan
   of the artificial joint used in the replacement procedure is often
   limited. Additionally, the onset of ONFH has been occurring at a
   younger age in recent years. Consequently, an increasing number of
   scholars have turned their attention to early-stage treatments for
   ONFH, such as tissue engineering and regenerative medicine. This
   approach, which aims to facilitate self-repair of tissues and reverse
   early-stage osteonecrosis, has become a hot topic in current research
   [[57]9,[58]10].

   Bone marrow mesenchymal stem cells (BMSCs) therapy, as a regenerative
   approach, has been increasingly utilized in research for treating
   early-stage ONFH. These cells possess the potential for self-renewal,
   multi-lineage differentiation, and immunomodulation. Furthermore, BMSCs
   exhibit excellent proliferative capacity, preserving their chromosomal
   karyotype and telomerase activity even after multiple passages
   [[59][11], [60][12], [61][13]]. The treatment of ONFH with BMSCs
   typically involves intra-bone marrow core decompression injection.
   However, this simple approach of solely injecting BMSCs often falls
   short of expected outcomes. Several factors may contribute to the
   suboptimal effects of direct BMSCs injection therapy, including
   mechanical damage to cells during the injection process, extensive
   infiltration of cells into surrounding tissue gaps leading to low cell
   retention rate, and compromised cell functionality due to inadequate
   extracellular matrix accumulation after injection. Therefore, a
   straightforward method to enhance the effectiveness of cell injection
   therapy is through high-dose multiple injections. This strategy,
   theoretically, can address the issues of low cell retention and
   survival rates [[62]11,[63]14]. However, the use of high-dose multiple
   cell injections increases treatment costs and reduces the practicality
   of cell therapy [[64]15]. Furthermore, high-dose cell injections may
   lead to excessive cell expansion and uncontrollable aberrant mutations,
   particularly when using genetically modified cells, thereby further
   increasing the associated risks [[65]14,[66]16]. Therefore, the
   exploration of an efficient and safe cell therapy has become a current
   research hotspot, with suitable cell carrier materials emerging as a
   promising choice. The objective is to achieve cell protection and
   minimize cell damage during the injection process, while increasing
   cell retention and survival rates, ultimately improving the efficacy of
   cell therapy [[67]17,[68]18]. Common cell carrier materials are often
   in the form of hydrogels. However, hydrogels alone are not effective in
   promoting extracellular matrix accumulation or facilitating
   interactions between cells [[69]16]. Therefore, we believe that ideal
   cell carrier materials should possess the following characteristics: 1)
   safety and non-toxicity with good cell compatibility; 2) injectability
   and biodegradability; 3) certain adsorption capacity to increase cell
   retention; 4) provide physical protection for cells; 5) create a
   physiologically relevant environment, such as a three-dimensional
   growth environment, to enhance the accumulation of extracellular
   mechanisms and interactions between cells. Ultimately, these properties
   improve cell utilization and prevent the need for high-dose repeated
   injections [[70][19], [71][20], [72][21], [73][22]].

   Therefore, our team has developed a 3D microscaffold material as a cell
   carrier. This microscaffold is stored in the form of tablets, which
   rapidly disperse into numerous microscaffold particles when dissolved
   in a neutral solution. Under a microscope, these microscaffold
   particles exhibit a three-dimensional structure with interconnected
   pores, providing a suitable environment for cell attachment and growth
   [[74][23], [75][24], [76][25]], while facilitating efficient transport
   of nutrients, oxygen and waste products. Instead of only increasing the
   surface area, as for traditional porous microspheres, our
   microscaffolds provide a 3D microenvironment that enables automatic and
   homogeneous loading of cells, matrices, and/or bioactive factors to
   construct tailored cellular niches. Our previous research has
   demonstrated that the microscaffold particles loaded with stem cells
   can enhance the therapeutic efficacy of cells. In a study using a rat
   model of knee osteoarthritis, we found that intra-articular injection
   of microscaffold-loaded low-dose stem cells produced similar
   therapeutic effects to the injection of high-dose stem cells alone
   [[77]16,[78]23].

   In this study, we for the first time utilized a 3D microscaffolds
   loaded with low-dose bone marrow mesenchymal stem cells for the
   treatment of steroid-induced femoral head necrosis in a rat model. We
   also established an in vitro co-culture system of osteoblasts,
   MPS-induced osteoblasts, pure BMSCs, and BMSCs grown in 3D
   microscaffolds for the analysis and identification of osteoblasts
   within the co-culture system. And through RNA-seq analysis and
   molecular biology validation, we have, for the first time,
   preliminarily explored the mechanisms underlying the therapeutic
   effects of the low-dose BMSCs-loaded 3D microscaffolds in femoral head
   necrosis. These findings confirm that the 3D microscaffolds loaded with
   low-dose bone marrow mesenchymal stem cells may be a promising
   therapeutic strategy for early-stage steroid-induced femoral head
   necrosis.

2. Materials and methods

2.1. Experimental design

   Human BMSCs were seeded in spinner culture flasks and co-cultured with
   3D microscaffolds for 4 days, resulting in the formation of a
   microscaffold suspension loaded with BMSCs. Subsequently, these
   suspensions were injected into the rat femoral head necrotic area. A
   co-culture system was employed to investigate the effects of 3D
   microscaffolds loaded with human BMSCs and free human BMSCs separately
   on human osteoblasts under both steroid-induced and non-steroid-induced
   conditions. The flowchart of experimental design is shown in [79]Fig.
   1.

Fig. 1.

   [80]Fig. 1
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   The flowchart of experimental design.

2.2. Isolation and cultivation of BMSCs from SD rats

   Isolation and cultivation of bone marrow mesenchymal stem cells from
   SPF-grade SD rats weighing 100–150g: The rats were anesthetized, and
   their bones were aseptically isolated and rinsed with sterile
   phosphate-buffered saline (PBS). The ends of the bones were cut using
   ophthalmic scissors to expose the bone marrow cavity. The bones were
   then placed in a sterile culture dish containing 10 ml of fresh DMEM
   complete medium supplemented with 10 % fetal bovine serum. A 1 ml
   syringe was used to flush the bone marrow cavity with complete medium,
   collecting the flush solution in another culture dish. This flushing
   step was repeated until the flush solution became clear. The bone
   marrow suspension was then centrifuged at 1000 r/min for 5 min at room
   temperature to remove the supernatant. The cell pellet was resuspended
   in 6 ml of complete medium to create a single-cell suspension, which
   was transferred to a 25 cm2 culture flask in a humidified incubator.
   After 24 h of culture, the medium was changed to remove floating blood
   cells. Subsequently, the medium was changed every two to three days
   until cell proliferation reached confluence [[82]26,[83]27]^.

2.3. Morphological observation of 3D microscaffolds

   The 3D microscaffolds, provided by School of Medicine, Tsinghua
   University, were stored in tablet form ([84]Fig. 2A) [[85]28]. One
   tablet of 3D microscaffolds was dissolved in phosphate-buffered saline
   (PBS), and after complete dissolution and dispersion, the mixture was
   centrifuged to remove the supernatant. The 3D microscaffolds were then
   sputter-coated with gold for 90 s and imaged using scanning electron
   microscopy (SEM; HITACHI, Japan).

Fig. 2.

   [86]Fig. 2
   [87]Open in a new tab

   (A) 3D culture of BMSCs. (B) Packed 3D microscaffolds in a tablet form.
   (C) Morphology of 3D microscaffolds dispersed in culture medium. (D)
   Fluorescent images of live/dead staining of BMSCs on days 1, 4, 7,14
   and 21 of culture. (E1-E2) Electron microscopy image of 3D
   microscaffolds. (E3-E4) BMSCs adherently grown on 3D microscaffolds.
   (F1-F2) Femoral head of SD rats. (F1) Normal femoral head. (F2) Femoral
   head after 8 weeks of MPS intervention. (G1-G2) Intraoperative X-ray
   image of a rat. (G1) Anteroposterior radiograph. (G2) Lateral
   radiograph. The red arrow: The insertion of a Kirschner wire into the
   necrotic area along the femoral neck. (H) Relative gene expression of
   stemness-related markers (SOX2, OCT4, NANOG) and senescence-related
   markers (P21, P16, P53) in BMSC-laden 3D microscaffolds over 21 days of
   culture (n = 3). (I) Flowcytometry analysis of surface biomarker
   expression over 14 days (n = 1, corresponding to a pool of 3
   microscaffolds).

2.4. Preparation and characterization of BMSCs-laden 3D microscaffolds

2.4.1. Preparation of BMSCs-laden 3D microscaffolds using the spinner culture
flasks

   Five 3D microscaffolds (specifications: 20 mg per microscaffold) were
   placed into a sterile 125 ml spinner culture flasks. Then, 20 ml of
   specialized complete medium was added to the culture bottle to fully
   dissolve the microscaffolds. Next, 1 ml of rat BMSCs suspension
   containing 2.5 × 10^6 cells were slowly added along the side wall of
   the spinner culture flasks. After that, an additional 30 ml of
   specialized complete medium was added to the culture bottle. The
   initial cell culture system comprising 50 ml was placed on a magnetic
   stirrer in a cell culture incubator, with an interval start-up
   consisting of 35 rpm/min for 5 min followed by 0 rpm/min for 1 h for a
   total of 24 cycles. The cell culture incubator maintained an
   environment of 37 °C and 5 % CO[2]. On the following day, the speed of
   the spinner culture flasks was adjusted to a constant speed of
   40 rpm/min, and 25 ml of fresh complete medium was supplemented. The
   culturing process continued for 21 days, during which the medium was
   replaced based on the glucose concentration in the culture supernatant
   [[88]23,[89]25]^.

2.4.2. Observation of BMSCs viability in 3D microscaffolds (live/dead
staining)

   Viability staining of BMSCs in 3D microscaffolds in the culture system
   was performed on days 1, 3, 7, 14, and 21. According to the
   manufacturer's instructions, a 100 μL suspension of cells and
   microscaffolds was extracted from the culture system and added to a
   96-well plate. The supernatant was aspirated and Calcein AM and
   Propidium iodide (PI) (Wako, Japan) were added. The plate was then
   placed in a light-protected environment at room temperature for 20 min.
   Subsequently, the supernatant was discarded, and one PBS wash was
   performed. Finally, the stained samples were observed using a
   fluorescence microscope [[90]14,[91]16].

2.4.3. Quantitative RT-PCR of BMSCs in 3D microscaffolds

   Quantitative RT-PCR was carried out on BMSCs in 3D microscaffolds on
   days 1, 3, 7, and 14. For each sample, reverse transcription was
   performed using the ReverTra Ace Qrna RT Master Mix with gDNA Remover
   (TOYOBO). Quantitative RT-PCR (qRT-PCR) was performed using the CFX96
   Real-Time Quantitative PCR System (Bio-Rad, USA).

   The samples were denatured at 95 °C for 30 s and then subjected to 40
   cycles of amplification: initial denaturation at 95 °C for 1 min,
   denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, and
   extension at 72 °C for 30 s. Subsequently, fluorescence signals were
   collected and recorded to generate amplification curves and melt
   curves. Ct values were then determined. The Ct values of the products
   were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as
   an internal control, and the relative expression levels of the gene
   were calculated using the 2–ΔΔCT method.

2.4.4. Flow cytometry of BMSCs in 3D microscaffolds

   SD rats’ BMSCs were stained with antibodies against MSC markers CD73,
   CD90, and CD105, as well as negative markers CD34 and CD45. Unstained
   BMSCs were used as negative controls. The cells were analyzed by flow
   cytometry (BD LSRFortessa, US).

2.5. Co-culture model of human osteoblasts and human BMSCs in femoral head
necrosis

2.5.1. Isolation and culture of human osteoblasts

   Primary human osteoblasts were isolated from femoral heads of patients
   with steroid-induced femoral head necrosis requiring total hip
   arthroplasty (THA). All patients provided informed consent, and the
   study was approved by the Ethics Committee of the 920th Hospital of the
   Chinese People's Liberation Army Joint Logistic Support Force.

   Inclusion criteria for patients were as follows: 1) End-stage primary
   steroid-induced femoral head necrosis. Exclusion criteria were: 1)
   Other types of femoral head necrosis. 2) Patients with underlying
   medical conditions that precluded surgery, such as diabetes, heart
   disease, etc.

   The inclusion criteria for patient selection were: 1) End-stage primary
   steroid-induced femoral head necrosis. The exclusion criteria were: 1)
   Other types of femoral head necrosis, 2) Patients with underlying
   medical conditions that precluded surgery, such as diabetes, heart
   disease, etc. Femoral head specimens were obtained and carefully
   dissected to remove periosteum and soft tissues. The specimens were
   then trimmed into 1mm3 bone blocks and placed in culture dishes
   containing DMEM/F12 medium. The bone blocks were transferred to EP
   tubes containing 2–3 mL of PBS, vigorously shaken, allowed to settle
   for 30 s, and the supernatant was removed. This process was repeated 3
   times using sterile PBS for rinsing. Subsequently, the bone blocks were
   treated with a red blood cell lysis buffer containing sodium
   bicarbonate (840 mg), ethylenediaminetetraacetic acid (EDTA, 37.2 mg),
   ammonium chloride (8.023 g), and double-distilled water (1000 ml) at a
   pH of 7.2 and a volume ratio of 1:3. The mixture was incubated for
   8 min, centrifuged, and the supernatant was discarded. The bone blocks
   were placed in centrifuge tubes until they turned white. Then, a
   10-fold volume of 2.5 g/L trypsin was added and pre-digested at 37 °C
   for 20 min in a constant temperature incubator. The digestion was
   terminated with fetal bovine serum, and the digestion solution was
   discarded. The bone blocks were washed twice with PBS. Subsequently, a
   0.1 % solution of type I collagenase at a volume ratio of 1:8 was
   added, and the tubes were sealed and placed in a 37 °C water bath for
   1 h of digestion with oscillation. After centrifugation at 1000 rpm for
   5 min, the supernatant was discarded. The precipitation was washed with
   PBS and centrifuged again at 1000 rpm for 5 min. After removing the
   supernatant, the precipitation was resuspended in complete DMEM-F12
   medium and pipetted to homogenize the cell suspension. The suspension
   was filtered through a sieve to remove non-osteoblastic cells and
   impurities, while retaining the bone particles. The remaining bone
   particles were subjected to three repeated digestions with type I
   collagenase. The cell suspension obtained from the three digestions was
   adjusted to a concentration of 5 × 105 cells/mL (as determined by
   trypan blue staining, with a cell viability of not less than 95 %) and
   seeded in sterile culture plates. The cells were cultured in a 37 °C,
   5 % CO[2] incubator. 48 h after seeding, the medium was changed, and
   the suspended cells were discarded. The medium was changed every 2 days
   until the cells approached confluence, which was considered as passage
   0 [[92]29].

2.5.2. Co-culture of human osteoblast and human BMSCs using transwell

   Primary isolated and identified human osteoblasts were cultured in
   α-MEM medium supplemented with 10 % fetal bovine serum and 1 % dual
   antibiotic (penicillin-streptomycin) and maintained at 37 °C with 5 %
   CO[2]. When the cell confluence reached 70 %, the cells were treated
   with an equal volume of either 0 μmol/L or 1 μmol/L methylprednisolone
   (MPS) as an intervention. The cells treated with 0 μmol/L MPS served as
   the control group, while those treated with 1 μmol/L MPS represented
   the steroid-induced necrosis of the femoral head (SINFH) model group.
   After a 24-h intervention period, the osteoblasts were collected for
   co-culture. A transwell system was utilized to establish the co-culture
   system, which was divided into two groups: a 2D co-culture group and a
   3D co-culture group. In the 2D co-culture group, the osteoblasts
   treated with 0 μmol/L intervention and the human osteoblasts treated
   with 1 μmol/L MPS were separately seeded at a density of
   5 × 10^5 cells/mL in the lower chamber of the transwell co-culture
   system, while the same number of free human BMSCs (1 × 10^6 cells) was
   seeded in the upper chamber of the transwell system. In the 3D
   co-culture group, human osteoblasts were seeded in the lower chamber of
   the transwell system using the same method, and 3D microscaffolds
   loaded with human BMSCs (1 × 10^5 cells) were seeded in the upper
   chamber. In both co-culture groups, the two cell types were not
   completely physically separated but allowed interaction only through
   diffusion factors. The co-culture systems were incubated in a 5 %
   CO[2], 37 °C culture incubator for 48–72 h, and then the human
   osteoblasts were collected for subsequent analysis [[93][30], [94][31],
   [95][32]].

2.5.3. The CCK-8 assay

   The osteoblasts from each co-culture group cultured for 48–72 h were
   collected for CCK-8 assay. CCK-8 reagent (10 μl per well) was added to
   the osteoblasts, and the culture plates were then incubated at 37 °C
   for 2 h inside the incubator. The optical density (OD) was measured at
   450 nm.

2.5.4. EdU imaging detection

   Osteoblasts from each co-culture group cultured for 48–72 h were
   collected for EdU proliferation staining. A 2X EdU working solution was
   prepared by diluting the final concentration of 10 μM EdU working
   solution with cell culture medium at a ratio of 1:500. Subsequently,
   20 μM of preheated 2X EdU working solution was added to each well of a
   6-well plate and the cells were incubated for an additional 2 h. After
   removing the fixative, cells were washed with 1 mL of wash buffer three
   times for 3–5 min each. The wash buffer was then removed, and 1 mL of
   permeabilization solution was added to each well, followed by
   incubation at room temperature for 10–15 min. After removing the
   permeabilization solution, cells were washed with 1 mL of wash buffer
   1–2 times for 3–5 min each. Subsequently, 0.5 mL of Click reaction
   solution was added to each well and the culture plate was gently shaken
   to ensure even coverage of the reaction mixture on the samples. The
   plate was then incubated at room temperature in the dark for 30 min.
   Finally, images were captured, and the percentage of positive cells was
   calculated.

2.5.5. Alkaline phosphatase activity assay was performed on human osteoblasts

   Alkaline phosphatase activity assay was performed on osteoblasts from
   each co-culture group cultured for 48–72 h. The collected osteoblasts
   were washed with PBS, fixed with ALP fixative for 3 min, and then
   rinsed with distilled water. Prepared ALP incubation solution (B1 and
   B2 diluted in a 1:1 ratio) was added dropwise to each group of
   osteoblasts. The samples were placed in a humid chamber and incubated
   in the dark for 15–20 min, followed by rinsing with distilled water.
   Subsequently, the samples were stained with nuclear Fast Red solution
   for 3–5 min, rinsed with PBS, and then examined under a microscope.

2.5.6. Flow cytometry analysis

   Osteoblasts from each group were washed twice with PBS and adjusted to
   a cell density of 1 × 10^6 cells/ml. A 1 mL cell suspension was
   centrifuged at 1300 rpm for 5 min, and the supernatant was discarded.
   After resuspending the cells in 500 μL of buffer, staining was
   performed according to the standard protocol of the Anexin-V FITC/PI
   Cell Apoptosis Detection Kit. Stained samples were acquired using the
   BD FACSCalibur flow cytometer, collecting data from 10^5 cells per
   sample. Each sample was measured three times, and the results were
   obtained using FlowJo software. The proportions of viable cells, early
   apoptotic cells, late apoptotic cells, and dead cells were
   statistically analyzed.

2.5.7. Quantitative RT-PCR

   Gene expression analysis of co-cultured osteoblasts in each group was
   conducted following the steps described in Section 2.2.3.

2.5.8. Western blotting

   The expression levels of Wnt1 and β-catenin proteins in co-cultured
   osteoblasts from each group were assessed using Western blot analysis.
   Cytosolic proteins were extracted directly using
   radioimmunoprecipitation assay (RIPA) lysis buffer (Servicebio)
   supplemented with a cocktail of protease and phosphatase inhibitors.
   The protein concentration was determined using the BCA Protein Assay
   Kit (Beyotime Biotechnology). Western blot analysis was conducted
   utilizing a kit according to the manufacturer's instructions, and
   chemiluminescent signals were generated using ECL reagent (Affinity).

2.5.9. Immunofluorescence taining

   Osteoblasts from each group were fixed in 4 % paraformaldehyde (PFA)
   for 15 min at room temperature, followed by permeabilization with 0.1 %
   Triton X-100 for 10 min. After blocking with 5 % bovine serum albumin
   (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature,
   the cells were incubated overnight at 4 °C with primary antibodies
   against osteocalcin (OCN) (Proteintech, America) and alkaline
   phosphatase (ALP) (ABclonal, America), each diluted at 1:200(OCN) and
   1:50 (ALP) in 1 % BSA/PBS. The following day, cells were washed three
   times with PBS and incubated with fluorescently labeled secondary
   antibodies (Donkey Anti-Mouse IgG H&L and Donkey Anti-Rabbit IgG H&L,
   Abcam, England) at a dilution of 1:400 for 1 h at room temperature in
   the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole
   (DAPI) (Abcam, England) for 5 min, followed by three washes with PBS.
   Fluorescent images were captured using a Zeiss LSM 710 confocal
   microscope under identical exposure settings. Quantification of
   fluorescence intensity was performed using ImageJ to ensure consistency
   across samples.

2.6. Animal experiments

2.6.1. Steroid-induced ONFH rat model

   The animal experiments were conducted in accordance with the guidelines
   set by the Institutional Animal Care and Use Committees (IACUC), and
   approval was obtained from the Ethics Committee of the 920th Hospital
   of the People's Liberation Army Joint Logistic Support Force (Approval
   No. 2022-039-01). A total of 56 male Sprague Dawley rats (8–10 weeks
   old and weighing 250–300 g) were purchased from the Animal Experiment
   Center of Kunming Medical University. The subjects were randomly
   divided into the following groups: Control (n = 8), Low
   BMSCs/microscaffolds (n = 8) (1 × 10^5 cell), High BMSCs(n = 8)
   (1 × 10^6 cell), Low BMSCs (n = 8) (1 × 10^5 cell), microscaffolds
   (n = 8) (0.5–0.7 mg), Core Decompression (CD)(n = 8), ONFH(n = 8).
   Except for the control group and ONFH model group, the remaining rat
   groups were administered intramuscular injections of methylprednisolone
   (MPS) (Pfizer, America). The dosage was 60 mg/kg once daily for 7
   consecutive days. Subsequently, all rats were carefully maintained for
   8 weeks [[96]33,[97]34]. Surgical procedure: After preparing the
   surgical area and inducing anesthesia through inhalation, the rats were
   positioned in a lateral recumbent position. The surgical site was
   disinfected and covered with surgical drapes. The center of the femoral
   head was located, and a longitudinal incision was made along the axis
   of the femoral neck to expose the underlying tissues. Using a 0.8 mm
   Kirschner needle, a needle was inserted at a 19^° angle, 20 mm from the
   knee joint, with a depth of approximately 5–6 mm. X-ray fluoroscopy was
   performed during the procedure to confirm successful placement of the
   puncture needle, as shown in [98]Fig. 2(E1-E2). Subsequently, the
   tunnel was rinsed with saline solution to remove bone debris. Injection
   treatments were administered according to the design requirements of
   each group. Finally, the tunnel opening was sealed with bone wax, and
   the incision was meticulously closed layer by layer.

2.6.2. Micro-CT analysis

   After 8 weeks of treatment, femoral heads were harvested and subjected
   to imaging scans using Micro-CT (SkyScan 1276, Germany, Bruker). The
   scanning parameters were set as follows: X-ray tube current of 200μA,
   voltage of 85 KV, scanning resolution of 10.141270 μm, exposure time of
   384 ms, and a scanning angle of 180° [[99]32]. The region of interest,
   which includes the subchondral bone 1 mm below the articular cartilage
   of the femoral head to the junction with the femoral neck, was scanned.
   Following the scan, the original images were reconstructed using
   three-dimensional reconstruction software NRecon (V1.7.4.2, Germany,
   Bruker), specifically selecting the region of interest. Subsequently,
   CT Analyser software (1.18.8.0, Germany, Bruker) was used to analyze
   the region of interest, including bone volume fraction (bone
   volume/total volume, BV/TV, %), trabecular spacing (Tb.Sp, mm),
   trabecular number (Tb.N, 1/mm), and trabecular thickness (Tb.Th, mm).

2.6.3. Histological and immunohistochemistry analysis

   The femoral head tissues of each rat group were fixed in a 4 %
   paraformaldehyde solution for 48 h. After removal, they were washed
   three times with PBS for 20 min each time, followed by three washes
   with distilled water for 20 min each time. Subsequently, the femoral
   head tissues were decalcified in a 10 % EDTA decalcification solution
   for a duration of 6 weeks, with the decalcification solution being
   replaced every 7 days. After completion of decalcification, the
   specimens were consecutively dehydrated, embedded in paraffin, and
   sectioned into 4 μm paraffin slices. The prepared paraffin sections
   underwent the following tests: Hematoxylin-eosin (H&E) staining for
   histological observations of tissue morphology under a light
   microscope; Masson staining for evaluation of collagen fiber synthesis
   in the femoral head tissues of each group under a light microscope;
   immunohistochemical (IHC) staining with markers including OCN
   (Osteocalcin) and Ki67 to assess the metabolic state of the femoral
   head tissues; Terminal-deoxynucleotidyl Transferase Mediated Nick End
   Labeling (TUNEL) staining for observation of cellular apoptosis in the
   femoral head tissues of each group under a fluorescence microscope.

2.6.4. RNA-seq analysis of rats’ femoral head

   The total RNA in the femoral head tissues of three rat groups, namely
   the Control group (group Z), Low BMSCs/microscaffolds group (group A),
   and ONFH group (group L), was isolated and purified using TRIzol
   reagent (Thermofisher, USA). Subsequently, the quantity and purity of
   the total RNA were assessed using a NanoDrop spectrophotometer
   (Nanodrop, USA), and the integrity of the RNA was evaluated using a
   bioanalyzer (Agilent, USA). The sequencing library was prepared using
   the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA)
   following the manufacturer's instructions. The reference genome index
   was generated using Hisat2 v2.0.5. Differential expression analysis was
   conducted between groups (with two biological replicates per condition)
   using the DESeq2 R package. All statistical analyses were performed
   using the R statistical programming language. Genes with an adjusted
   p-value of <0.05 as determined by DESeq2 were considered differentially
   expressed. Differentially expressed genes (DEGs) were defined as those
   exhibiting a fold change ≥2 and a p-value ≤0.05. Heatmaps were created
   using the heatmap package. Gene ontology (GO) enrichment analysis and
   KEGG pathway analysis were carried out using the cluster Profiler R
   package. For each group, three replicates were collected for RNA-seq
   analysis.

2.7. Statistical analysis

   Statistical analyses were performed using GraphPad Prism. All data are
   presented as mean ± standard deviation (SD). Student's t-test was used
   for comparing two groups, while one-way analysis of variance (ANOVA)
   followed by Tukey's post hoc test was used for comparing three or more
   groups. A value of P < 0.05 was considered statistically significant.
   All experiments were conducted using a minimum of three independent
   samples.

3. Results

3.1. Preparation and characterization of BMSCs-laden 3D microscaffolds

3.1.1. Morphological characteristics of 3D microscaffolds

   The 3D culture of BMSCs is shown in [100]Fig. 2A. The 3D microscaffolds
   are stored in the form of tablets and disperse into numerous individual
   microscaffolds particles when dissolved in neutral solutions such as
   physiological saline, culture media, or PBS ([101]Fig. 2B and C). The
   scanning electron microscopy image is shown in [102]Fig. 2(E1-E2).
   These microscaffolds particles exhibit a relatively uniform spherical
   or spheroid shape, with a diameter of 200 ± 7.35 μm. The microscaffolds
   particles are interconnected by numerous large pore structures,
   enabling cell adhesion and growth within these pore structures
   ([103]Fig. 2(E3-E4)). Image analysis using ImageJ revealed an average
   pore diameter of 17.78 ± 6.3 μm in these microscaffolds particles.

3.1.2. Live/dead staining

   Live/dead staining with Calcein AM/PI demonstrated abundant cell
   viability in the cells cultured in spinner culture flasks for 21 days,
   as shown in [104]Fig. 2D. A comprehensive cell counts of 1.26 × 10^7
   BMSCs was conducted on day 21 in the spinner culture flasks. To ensure
   both the quality and quantity of cells, we employed cells cultured in
   spinner culture flasks up to day 4 for animal experiments.

3.1.3. Quantitative RT-PCR of BMSCs in 3D microscaffolds

   The BMSCs in 3D microscaffolds were evaluated for stemness and
   senescence related gene markers by qRT-PCR. The results indicated that
   the expression levels of stemness-related markers SOX2, OCT4, and NANOG
   were higher at day 4, day 7, and day 14 compared to other time points.
   In contrast, the senescence-related markers P21, P16, and P53 showed
   significant downregulation after 3 days, as shown in [105]Fig. 2H. The
   collective gene expression results suggested that BMSCs maintained
   their stemness and suppressed senescence after being cultured in
   spinner flasks for 7–14 days. It is expected that similar effects will
   persist in vivo for at least 1–2 weeks after injection of 3D
   microscaffolds loaded with BMSCs pre-cultured until day 4.

3.1.4. Flow cytometry of BMSCs in 3D microscaffolds

   Flow cytometry analysis indicated that during the 14 days 3D culture
   period, BMSCs within the 3D microscaffolds maintained their original
   immunophenotype, with high expression of BMSC markers CD73, CD90, and
   CD105, and low expression of negative markers CD34 and CD45, as shown
   in [106]Fig. 2I. This demonstrates that the 3D microscaffolds do not
   alter the original immunophenotype of BMSCs, thereby providing evidence
   for the safety of cell culture using 3D microscaffolds to a certain
   extent.

3.2. Effects of 3D microscaffolds loaded with BMSCs on a steroid-induced
femoral head necrosis model in SD rats

   All rats survived for 8 weeks following intramuscular injection of MPS,
   and subsequent anatomical examinations revealed the presence of femoral
   head necrosis in all cases, as depicted in [107]Fig. 2(F1) and (F2).

3.2.1. μ-CT analyses

   μ-CT analysis reveals alterations in trabecular bone architecture in
   the subchondral region of the femoral head in rats. As shown in
   [108]Fig. 3A, the control group exhibited normal trabecular bone
   density, while the ONFH group displayed sparse trabecular arrangement
   and erosive changes in the medullary bone. The Low BMSCs/microscaffolds
   group and High BMSCs group exhibited denser trabecular arrangement
   compared to the control group. As shown in [109]Fig. 3D-G, bone
   analysis software analysis demonstrated significantly increased BV/TV,
   Tb.N, and Tb.Th, as well as decreased Tb. Sp in the Low
   BMSCs/microscaffolds group and High BMSCs group when compared to the
   ONFH group, Low BMSCs group, microscaffolds group, and CD group. These
   findings suggest that 3D microscaffolds loaded with low-dose BMSCs
   effectively improve the reduced bone density induced by glucocorticoids
   and exhibit similar therapeutic effects to high-dose BMSCs
   administration.

Fig. 3.

   [110]Fig. 3
   [111]Open in a new tab

   (A) Images of μ-CT. (B–C) Histological (Hematoxylin & Eosin staining
   and Masson staining). The yellow arrow indicates increased adipocytes.
   (D–G) Quantitative analysis of BV/TV, Tb.N, Tb.Th and Tb. Sp in the
   femoral head. (H)The empty lacunae percentages of each group. (I) The
   collagen fiber proliferation.

3.2.2. Histological and immunohistochemistry analysis

   Histopathological observations of femoral head necrosis using H&E
   staining. Results revealed that the control group exhibited normal
   histological features of femoral head tissue, with intact articular
   cartilage structure, regular cellular arrangement, absence of fissures,
   well-aligned trabecular pattern, uniform distribution of bone cells,
   and normal hematopoietic tissue distribution in the bone marrow cavity,
   without evident empty lacunae or increased adipocytes. In contrast, the
   ONFH group showed evident signs of femoral head necrosis, including
   reduced number of chondrocytes in the articular cartilage, nuclear
   shrinkage, decreased cell count, significantly thinner and even
   fractured trabeculae with sparse and reduced distribution, marked
   increase in red blood cell count, and noticeably elevated numbers of
   empty lacunae and adipocytes. Dense Trabecular Arrangement in the Low
   BMSCs/microscaffolds Group and High BMSCs Group compared to the ONFH
   Group, with reduced empty lacunae and adipocytes compared to the ONFH
   Group, as shown in [112]Fig. 3B. The empty lacunae percentages for each
   group are presented in [113]Fig. 3H. In conclusion, 3D microscaffolds
   loaded with low-dose BMSCs and high-dose BMSCs injection both exhibit
   similar effects on femoral head necrosis. Masson's staining results
   revealed minimal collagen fiber proliferation representing new bone
   tissue in the Control group. In comparison to the Control group, the
   ONFH group exhibited abundant red muscle fibers within the trabeculae,
   with no apparent increase in collagen fiber tissue. When comparing the
   Low BMSCs/microscaffolds group, High BMSCs group, ONFH group, and
   Control group, both the Low BMSCs/microscaffolds group and the High
   BMSCs group demonstrated significant collagen fiber proliferation,
   surpassing the other treatment groups, as shown in [114]Fig. 3C and I.
   TUNEL staining was performed to observe cellular apoptosis in the rat
   femoral head tissue. The positive cells were labeled with red
   fluorescent staining on the sections. The overlay images of Tunel and
   DAPI staining revealed pink-colored positive cells. The apoptosis index
   was determined by randomly capturing 5 fields of view under high
   magnification on the same section and calculating the average ratio of
   positive cells in these 5 fields. Compared to the Control group, the
   ONFH group showed a significant increase in the apoptosis index
   (p < 0.01). In comparison, the Low BMSCs/microscaffolds group and the
   High BMSCs group exhibited a significant reduction in the apoptosis
   index (p < 0.0001), which was lower than other treatment groups. The
   results are shown in [115]Fig. 4A and C. Immunohistochemical staining
   was conducted to evaluate the proliferation of osteoblasts in the rat
   femoral head tissue. Compared to the control group, the ONFH group
   exhibited a significant decrease in the expression level of osteocalcin
   (OCN) (p < 0.001). In comparison, both the Low BMSCs/microscaffolds
   group and the High BMSCs group demonstrated a significant increase in
   OCN-positive cells compared to the ONFH group (p < 0.001), surpassing
   other treatment groups. The findings are presented in [116]Fig. 4B and
   D. Similarly, when compared to the Control group, the ONFH group
   displayed a significant decrease in the expression level of nuclear
   protein Ki67 (p < 0.01). In contrast, both the Low BMSCs/microscaffolds
   group and the High BMSCs group exhibited a significant increase in the
   percentage of Ki67-positive cells compared to the ONFH group
   (p < 0.0001), surpassing other treatment groups. The results are
   depicted in [117]Fig. 4B and E.

Fig. 4.

   [118]Fig. 4
   [119]Open in a new tab

   (A) Tunel and DAPI staining. (B) Immunohistochemical staining (OCN and
   Ki67). The black arrow: OCN positive cells. The red arrow: Ki67
   positive cells. (C) TUNEL positive cells percentages. (D–E) OCN
   positive cells percentages and Ki67 positive cells percentages of each
   group.

3.3. In vitro co-culture of human bone marrow mesenchymal stem cells and
human osteoblasts

   Co-culture of human bone marrow mesenchymal stem cells and human
   osteoblasts was performed using Transwell inserts. Subsequently, the
   effects of 3D microscaffolds loaded with BMSCs and free BMSCs on human
   osteoblasts were evaluated through analysis.

3.3.1. CCK-8 assay

   The CCK-8 assay results demonstrated that the osteoblasts in the 3D
   group exhibited significantly higher OD values compared to the 2D
   group, both in MPS-induced and non-induced environments ([120]Fig. 5E),
   indicating statistically significant differences (p < 0.0001). This
   suggests that BMSCs cultured on 3D microscaffolds have a better ability
   to accumulate cell matrix, thereby promoting osteoblast vitality.

Fig. 5.

   [121]Fig. 5
   [122]Open in a new tab

   (A) EdU staining of osteoblasts in 2D groups and 3D groups. (B) ALP
   staining of osteoblasts in 2D groups and 3D groups. (C) Apoptotic
   status of osteoblasts in different groups. Flow cytometry analysis
   results of osteoblasts. (D) Expression profile of Wnt1 and β-Catenin
   proteins. (E) CCK-8 assay. OD values of different groups. (F) EdU
   positive cell rates of different groups. (G) ALP positive cell rates of
   different groups. (H) Percentage of positive apoptotic cells in
   different groups of osteoblasts. (I–J) Grayscale intensity of Wnt1 and
   β-Catenin protein expression.

3.3.2. EdU imaging assay

   The EdU imaging results revealed that the osteoblast positivity rate in
   the 3D group was significantly higher than that in the 2D group, both
   in MPS -induced and non-induced environments, with statistically
   significant differences ([123]Fig. 5A and F). This indicates that BMSCs
   cultured on 3D microscaffolds have a greater ability to promote
   osteoblast proliferation.

3.3.3. Alkaline phosphatase (ALP) assay

   The ALP detection results revealed a significantly higher percentage of
   ALP-positive osteoblasts in the 3D group, both in the presence and
   absence of MPS-induction, compared to the 2D group. These differences
   were statistically significant, as shown in [124]Fig. 5B and G. This
   indicates that BMSCs cultured on 3D microscaffolds demonstrated
   enhanced expression of alkaline phosphatase by osteoblasts, thereby
   promoting osteoblast activity more effectively.

3.3.4. Flow cytometry analysis

   Flow cytometry analysis revealed significantly lower percentages of
   early apoptosis, late apoptosis, and dead cells in the 3D group of
   osteoblasts compared to the 2D group, both in the presence and absence
   of MPS-induction. These differences were statistically significant, as
   illustrated in [125]Fig. 5C and H. These results suggest that BMSCs
   cultured on 3D microscaffolds exhibit enhanced maintenance of
   osteoblast activity, leading to reduced cellular apoptosis.

3.3.5. Western blotting

   The Western blot analysis revealed significantly higher relative
   expression levels of Wnt1 and β-catenin proteins in the 3D group
   compared to the 2D group, both in the presence and absence of
   MPS-induction. These differences were statistically significant, as
   depicted in [126]Fig. 5D, I and 5J. These findings indicate that BMSCs
   cultured on 3D microscaffolds can significantly activate the
   Wnt1/β-catenin pathway, promoting the relative expression levels of
   Wnt1 and β-catenin proteins, accelerating tissue repair in the necrotic
   area, and improving the condition of femoral head necrosis.

3.3.6. Immunofluorescence staining

   Immunofluorescence staining demonstrated that the relative fluorescence
   density of OCN and ALP in the 3D group were significantly higher than
   those in the 2D group, irrespective of MPS induction ([127]Fig. 6A).
   These differences were statistically significant, as shown in [128]Fig.
   6B and C. These findings indicate that culturing bone marrow
   mesenchymal stem cells on 3D microscaffolds enhances the expression of
   OCN and ALP in osteogenic cells, thereby promoting the repair of
   femoral head necrosis.

Fig. 6.

   [129]Fig. 6
   [130]Open in a new tab

   (A) OCN and ALP immunofluorescence staining of each group. (B–C)
   Semi-quantitative analysis of OCN and ALP immunofluorescence intensity
   (n = 3). Statistical analysis was performed using an ANOVA with Tukey's
   post hoc test. Statistical significance was set at P < 0.05.

3.4. RNA-seq analysis of rats’ femoral head

   To gain insights into that early osteonecrosis of the femoral head was
   alleviated by cell therapy of low-dose BMSCs-loaded 3D microscaffold,
   the potential mechanisms were explored with bulk-RNA sequencing to
   identify the differentially expressed genes (DEGs) in the femoral head
   samples of each group. The samples were clearly separated into 3
   expression patterns by a heatmap analysis of all identified expressed
   genes ([131]Fig. 7A). Volcano plots showed 4379 DEGs between the group
   A and the group L, while 3807 DEGs were identified between the group L
   and the group Z ([132]Fig. 7B).

Fig. 7.

   [133]Fig. 7
   [134]Open in a new tab

   (A) Differential gene clustering plot of Group Z, Group A and Group L.
   (B) Differential gene volcano plot of Group A vs Group L and Group L vs
   Group Z. (C) Heatmap of gene expression differences between group A and
   group L in the MAPK signaling pathway and Hippo signaling pathway. (D)
   Signal pathway diagram of MAPK signaling pathway. (E) Signal pathway
   diagram of Hippo signaling pathway. (F) Expression of TGFBR2, MPK2A6
   and HSPBl protein in MAPK signaling pathway in each group of samples.
   (G) Expression of YAP1, TEAD and CCNDl protein in Hippo signaling
   pathway in each group of samples. (H–I) Statistical analysis of each
   protein expression (n = 3).

   KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment
   analysis was conducted to assess the functions of these DEGs in
   biological functions. In comparison to the L group, several processes
   were found to be significantly upregulated in the A Group, Including
   MAPK signaling pathway, Hippo signaling pathway - multiple species,
   Calcium Signaling Pathway, Focal Adhesion, PI3K-Akt Signaling Pathway,
   Rap1 Signaling Pathway, Regulation of Actin Cytoskeleton and so on
   ([135]Supplemental Table X1). In comparison to the Z group, several
   processes were found to be significantly down-regulated in the L Group,
   including ECM-Receptor Interaction, Hippo signaling pathway, MAPK
   signaling pathway, Rap1 Signaling Pathway, cGMP-PKG Signaling Pathway,
   Regulation of Actin Cytoskeleton, Cell Adhesion Molecules and so on
   ([136]Supplemental Table X2). Furthermore, 15 key pathways related to
   the proliferation and osteogenic differentiation were selected
   ([137]Supplemental Table X3) and analyzed for the regulation of gene
   expression, between and the A group and the L group, demonstrating that
   MAPK signaling pathway and Hippo signaling pathway were significantly
   up-regulated ([138]Supplemental Table X4). The mitogen-activated
   protein kinase (MAPK) pathway, also known as the RAS-RAF-MEK-ERK
   pathway, is a critical intracellular signaling cascade that facilitates
   the repair of femoral head necrosis by promoting osteogenic
   differentiation, enhancing angiogenesis, suppressing apoptosis, and
   modulating inflammatory responses. Similarly, the Hippo signaling
   pathway contributes to the repair process by regulating mesenchymal
   stem cell (MSC) differentiation, promoting vascularization, and
   maintaining bone homeostasis, thereby providing essential cellular and
   vascular support ([139]Fig. 7C–E).

   KEGG pathway enrichment analysis indicating that TGFBR2, TGFB3, MPK2A6,
   P38, HSPB1, HSPBA1s genes were up-regulated in MAPK signaling pathway,
   focusing on TGFBR2, MPK2A6, HSPB1; FRMD6, YAP1, LTS1_2, TEAD, CCND1
   genes were up-regulated in Hippo signaling pathway, focusing on YAP1,
   TEAD, CCND1 ([140]Supplemental Table X5). Therefore, on the basis of
   the sequencing results, we speculate that MAPK signaling pathway and
   Hippo signaling pathway may be activated during the low-dose
   BMSCs-loaded 3D microscaffolds treatment of ONFH, these two pathways do
   not function in isolation; instead, they act synergistically to
   regulate key processes such as osteogenic differentiation,
   angiogenesis, and bone homeostasis, collectively driving the
   regeneration and repair of necrotic bone tissue. The rats’ femoral head
   tissues were analyzed again to test this hypothesis. Western blot
   analysis revealed that the expression levels of TGFBR2, MPK2A6, and
   HSPB1 in the MAPK signaling pathway were significantly elevated in
   group A compared to group L ([141]Fig. 7F). Similarly, in the Hippo
   signaling pathway, the expression levels of YAP1, TEAD, and CCND1 were
   significantly elevated in group A than in group L ([142]Fig. 7G). The
   statistical analyses are presented in [143]Fig. 7H and I.

4. Discussion

   In this study, we utilized an SD rat MPS-induced femoral head necrosis
   model and an in vitro co-culture model to investigate the therapeutic
   effect of 3D microscaffolds loaded with BMSCs on MPS-induced femoral
   head necrosis. Clinical evidence currently suggests that repeated
   high-dose injections of stem cell therapy demonstrate better treatment
   outcomes compared to single injections, although they also carry higher
   risks of infection and other complications [[144]35]. In animal
   experiments and in vitro co-culture studies, we have demonstrated,
   using methods such as Micro-CT, histology, and immunohistochemistry,
   that injection of 3D microscaffolds loaded with low-dose BMSCs exhibits
   similar therapeutic effects to high-dose BMSCs administered alone.
   Furthermore, through high-throughput sequencing of rat femoral head
   tissues, we have revealed the underlying molecular mechanisms of 3D
   microscaffolds loaded with low-dose BMSCs in the treatment of ONFH.

   In recent years, regenerative medicine has rapidly advanced, and the
   integration of stem cells with biomaterials has emerged as a
   fundamental strategy for treating various diseases [[145]36].
   Mesenchymal stem cells (MSCs) from various tissue sources are
   considered reliable cell types for tissue repair due to their high
   proliferative capacity and multipotency [[146]37,[147]38]. Among them,
   bone marrow derived MSCs (BMSCs) have gained increasing attention in
   the field of orthopedics due to their low immunogenicity, ease of
   culture and expansion, and multilineage differentiation potential.
   While it is generally believed that stem cell therapy is dose-dependent
   and that a higher cell quantity leads to greater efficacy, further
   high-quality evidence is necessary to establish the long-term safety
   and clinical advantages of high-dose stem cell injections
   [[148]39,[149]40]. It is worth noting that stem cells frequently
   undergo excessive expansion in two-dimensional (2D) culture, leading to
   a loss of their stemness and phenotypic characteristics. Therefore, the
   use of 2D culture methods for cell mass production is not an ideal
   approach [[150]41,[151]42]. It is well known that 3D culture provides
   stem cells with the ability to establish biomimetic hierarchical
   structures, thereby better maintaining their activity
   [[152]40,[153]43]. However, a safe, stable, and efficient method for 3D
   culture of stem cells and achieving mass production has been a
   challenge. In this study, the novel cell carrier material, 3D
   microscaffolds, which we used, offers a potential cell delivery
   platform to address this challenge. Numerous studies have shown
   significant advantages of 3D microscaffolds compared to other existing
   cell carrier materials [[154]24]. The microscaffolds used in this study
   are made from clinically applicable gelatin, which provides inherent
   cell adhesion motifs. Their 3D porous structure offers a natural growth
   microenvironment for BMSCs, thereby enhancing the retention rate and
   functionality of injected cells post-injection [[155]16]. In this
   study, we employed 3D microscaffolds for the extended 14-day 3D
   cultivation of BMSCs. Subsequently, we substantiated the safety and
   efficacy of BMSCs loaded onto 3D microscaffolds through live/dead
   staining, quantitative RT-PCR, and flow cytometry analysis.

   In our experimental study, we induced ONFH by intramuscular injection
   of glucocorticoids in rats. This model clearly establishes a
   relationship between glucocorticoids and the occurrence of ONFH
   [[156]34]. At the 8th week after glucocorticoid injection, histological
   examination revealed ONFH in all rats. We conducted corresponding
   therapeutic interventions on the rats according to a pre-designed
   grouping. Through imaging, histology, and immunohistochemistry
   analysis, we found that, at 8 weeks post-intervention, the injection of
   3D microscaffolds loaded with low-dose BMSCs showed similar therapeutic
   effects in repairing ONFH compared to the injection of high-dose BMSCs
   alone. Furthermore, both methods exhibited better therapeutic outcomes
   than the other treatment groups. Due to the limited source of primary
   BMSCs and the high cost of cell expansion culture, large-scale
   production of BMSCs for clinical application may not be economically or
   practically feasible. It is worth noting that stem cell injection
   therapy is more suitable for early-stage ONFH without obvious collapse,
   while its therapeutic efficacy is often poor for late-stage ONFH
   classified as Arco stages III and IV. In the early stages of ONFH, the
   synovial fluid can provide nourishment to the femoral head, and there
   may be inherent repair mechanisms. As a result, there are no apparent
   changes in articular cartilage. If early-stage ONFH is not promptly
   repaired, it will gradually progress into typical ischemic necrosis,
   where joint replacement surgery becomes the only effective treatment
   method [[157]44].

   Cell-cell interactions encompass a myriad of biological phenomena,
   including embryonic development, tissue morphogenesis, and repair
   [[158]45,[159]46]. Establishing a co-culture model of BMSCs and primary
   osteoblasts to simulate the physiological or pathological environment
   in vivo is an ideal research approach for investigating cell-cell
   interactions and evaluating therapeutic efficacy. In this model, BMSCs
   are surrounded by osteoblasts but do not directly contact them
   [[160]32]. In this study, we established a co-culture model of human
   osteoblasts and human BMSCs using Transwell and analyzed the
   osteoblasts in the model. Flow cytometry analysis and protein
   expression results demonstrated that BMSCs grown in 3D microscaffolds,
   both in the physiological and pathological environments, could better
   enhance osteoblast activity and maintain their phenotype. These
   findings suggest that 3D microscaffolds loaded with BMSCs may
   potentially facilitate bone repair in femoral head osteonecrosis more
   effectively. As a cell delivery vehicle, if these BMSC-loaded
   microscaffolds are applied clinically, we speculate that BMSCs
   primarily exert their therapeutic effects on femoral head osteonecrosis
   repair through cell matrix accumulation and paracrine function.
   Additionally, the use of 3D microscaffolds may minimize mechanical
   damage to cells during the injection process, thereby improving the
   retention and viability of BMSCs in vivo.

   Furthermore, this study has certain limitations. In our co-culture
   model of human BMSCs and human osteoblasts, we only investigated their
   phenotypic aspects and preliminary molecular mechanisms. Although we
   have demonstrated the superior promoting effect of 3D microscaffolds
   loaded with BMSCs on osteoblasts through these investigations, the
   intricate mechanisms underlying cell-cell interactions have not been
   elucidated. Future studies will focus on analyzing these molecular
   mechanisms. Additionally, our in vivo experiments were conducted on
   small rodent models, which may not fully reflect the reparative effects
   in humans. Subsequent research will be carried out on larger primate
   models to provide high-quality evidence supporting the clinical
   application of 3D microscaffolds loaded with low-dose BMSCs for
   early-stage femoral head osteonecrosis treatment.

5. Conclusion

   The treatment of early-stage steroid-induced femoral head osteonecrosis
   using a single injection of 3D microscaffolds loaded with low-dose
   BMSCs has achieved therapeutic outcomes similar to those achieved with
   the injection of high-dose free BMSCs. This clearly demonstrates the
   advantages of this treatment approach, as it significantly reduces the
   number of required cells and opens up possibilities for large-scale
   clinical applications. Additionally, this study represents the first
   exploration of the therapeutic effects of 3D microscaffolds loaded with
   BMSCs for early-stage steroid-induced femoral head osteonecrosis, and
   provides a preliminary investigation into the underlying molecular
   mechanisms, providing a promising treatment strategy for the
   hip-preserving therapy of femoral head osteonecrosis.

CRediT authorship contribution statement

   Minzheng Guo: Writing – original draft, Supervision, Resources,
   Methodology, Funding acquisition, Formal analysis, Data curation,
   Conceptualization. Baochuang Qi: Writing – review & editing, Software,
   Methodology, Investigation, Data curation, Conceptualization. Zijie
   Pei: Validation, Software, Methodology, Investigation. Haonan Ni:
   Software. Junxiao Ren: Software. Huan Luo: Software, Methodology.
   Hongxin Shi: Validation, Software. Chen Meng: Data curation,
   Validation. Yang Yu: Software, Methodology. Zhifang Tang: Software.
   Yongqing Xu: Writing – review & editing, Supervision, Resources,
   Project administration, Investigation, Funding acquisition, Formal
   analysis, Conceptualization. Qingyun Xue: Writing – review & editing,
   Supervision, Resources, Investigation, Data curation. Chuan Li: Writing
   – review & editing, Supervision, Resources, Project administration,
   Investigation, Funding acquisition, Data curation, Conceptualization.

Ethics approval and consent to participate

   The experimental protocol has been approved by the Ethics Committee of
   the 920th Hospital of the Chinese People's Liberation Army Joint
   Logistic Support Force (LS-2022-039(K)-01).

Consent for publication

   Not applicable.

Availability of data and materials

   A Data Availability statement is not applicable to this manuscript.

Declaration of AI and AI-assisted technologies in the writing process

   No AI or AI-assisted technologies were used in the writing or editing
   of this manuscript.

Funding

   This work was jointly supported by the Grants from Yunnan Orthopedics
   and Sports Rehabilitation Clinical Medicine Research Center Fund (grant
   numbers 202102AA310068), Yunnan Province Clinical Orthopaedic Trauma
   Medical Center Fund (grant numbers ZX20191001), Yunnan Province Science
   and Technology Talents and Platform Program Project Fund (grant numbers
   202105AD160027), Meiluo Young Physicians' Innovative Development
   Project Fund (grant numbers GSKQNJJ-2023-005).

Declaration of competing interest

   The authors declare no conflict of interest.

Acknowledgements