Tibial cortex transverse transport potentiates diabetic wound healing via activation of SDF-1/CXCR4 signaling

Diabetic rats

A total of 54 male SD rats (age, 6-weeks; weight range, 280–410 g) were used to establish the diabetic rat model. Diabetes was induced by intraperitoneal injection of 35 mg/kg STZ (YuanYe Bio, Shanghai, China) as described in a previous report (Furman, 2021). The rats were allowed acclimate to their new environment for at least 5 weeks prior to being injected with STZ. The rats were housed (five rats per cage) in a room maintained at 21  ± 1 °C, 55  ± 5% humidity, and with a 12-h light dark cycle (08.00 on: 20.00 off). The rats had ad libitum access to water and a high fat diet (formula details: 10% lard, 2.5% cholesterol, 20% sucrose, 1% cholate, and 66.5% common diet, respectively), for at least one week; after which they were fasted for 12 h prior to being injected with STZ. On the last day, after being fasted for 12 h, a sample of blood was obtained from the caudal vein of each rat and used to measure the blood glucose level. A blood glucose level >16.7 mmol/L indicated that the diabetic rat model was successfully established (Fig. 1A). The rats were obtained from the Animal Center of Southern Medical University and the protocols for all animal studies were approved by an Institutional Review Committee for the use of Animal Subjects, and conformed with guidelines established by the Second People’s Hospital of Guangdong Province Experimental Animal Ethics Committee (No. 2022-dw-kz-006-01).

Surgical procedures

The diabetic rats were allowed to recover for at least one week prior to being used in any experiments. All experimental rats were anesthetized and operated in a sterile operating room to minimize postoperative complications. The rats with successful diabetes modeling were divided into three groups: a sham operation group, tibial osteotomy group, and TTT group, respectively. A total of 18 diabetic rats that were not implanted or received a sham operation served as a negative control group, designated as the blank group. The tibial osteotomy group consisted of 18 diabetic rats that underwent fixator implantaton surgery; that group was designated as the fixator group. A total of 18 diabetic rats implanted with the TTT fixator formed the TTT group. The TTT procedure was performed as described in a previous study (Chen et al., 2021a; Furman, 2021) (Fig. 1B 1–6). The TTT procedure for rats required nine days to complete, and. the middle and upper segments of the tibia were selected for cortical osteotomy. Guided with the external fixator, a pneumatic saw was used to penetrate the bone cortex to form a corticotomy window of 10 mm × five mms. After punching holes, steel needles were passed through the cortex, fixed in the holes and then assembled to form an external fixator. Two 2-mm threaded needles were used to perform cortical bone chip transport. After surgery, the animals were allowed to rest for 3 days. On day 4 after surgery, the bone chip was transversely pulled outward by 0.25 mm every 12 h for three days, and then pulled inward in the same manner during the last three days. All rats were operated on the dorsum of the foot to form a full-thickness skin wound of four mm × 5 mm (Fig. 1B 7–8), No obvious signs of infection or other complications were observed in any of the experimental animals after the operation. The surgery day was designated as day 0. On the third day, the postoperative extension was extended for 5 days, stopped for 2 days, and then pulled back for 5 days in the TTT group. All 6 diabetic rats in each group were euthanized using a CO₂ euthanasia device (BM Shanghai Biowill Co., LTD., Shanghai, China) with a flow of 6.5 L/min on days 9, 16, and 21 after surgery, respectively. Changes in body weight were recorded, pictures of skin lesions were taken, and pathological examinations were performed (Fig. 1C).

Body weight

As mentioned above, all rats were subjected to uniform rearing standards. To limit variations in baseline data, body weights were recorded prior to injection of STZ, and diabetic rats were weighed a second time following successful modeling. Each rat was weighed prior to the procedure, and after the procedure, weight changes in the groups were monitored and recorded.

Skin wound recovery

Following surgery, the experimental rats were fed under normal conditions, and the ulcer wounds were photographed at various intervals.

Hematoxylin and eosin (H&E) staining

Morphological changes in skin wound tissue of the rat dorsal foot wounds were detected by H&E staining on days 9, 15, and 21 after surgery. The paraformaldehyde-fixed skin tissues were placed in a descending alcohol series; after which, they were dehydrated, fixed, and paraffin-embedded. The blocks of tissue were then cut into 3 µm-thick sections with a microtome. Next, the sections were deparaffinized, rehydrated, and immersed in hematoxylin for 5-8 min before being washed with phosphate-buffered saline (PBS). The sections were then immersed in 0.1% acid water and subsequently exposed to 1% eosin for 1-3 min. Finally, the sections were treated with an ascending alcohol series, cleared with xylene, mounted onto slides, and observed and photographed under a light microscope (NIKON, ECLIPSE Ci, Japan).

Immunofluorescence (IF)

As noted above, the prepared skin sections were deparaffinized and rehydrated prior to antigen retrieval. Following rehydration, the sections were placed in an enclosed plastic container, rinsed three times in 100 mL PBS for 5 min, and then subjected to antigen retrieval with EDTA (pH 8.0). A circle was draw around the tissues with an IHC PAP pen. The sections were blocked with blocking buffer for at least 30 mins at room temperature after antigen retrieval. The sections were then removed from the blocking buffer, treated with diluted primary antibodies, and incubated overnight in a wet chamber at 4 °C. The next morning, the tissues were incubated with a secondary antibody for 50 mins at room temperature. Finally, the nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; 0.1 µg/mL). The antibodies used in the study were anti-CD34 (sc-18917,1:200; Santa Cruz Biotechnology, Dallas TX, USA), anti-CD133 (sc-365537, 1:200; Santa Cruz Biotechnology), anti-CXCR4 (ab124824; Abcam, Cambridge, MA, USA, 1:200), anti-SDF-1 (17402-1-AP; Proteintech, Rosemont, IL, USA, 1:500), Goat anti-rat-IgG H&L (FITC, ab6840, 1:1,000; FITC, Abcam), Goat anti-mouse-IgG H&L (Alexa Fluor®594, ab150120, 1:200; Abcam), and Goat anti-rabbit IgG H&L (Cy5®, ab6564, 1000; Abcam).

Immunohistochemistry (IHC)

Sections of skin tissue were blocked with 3% H₂O₂ for 10 min, washed three times in PBS, permeabilized with 0.2%Triton for 10 min, and then incubated overnight with primary antibodies at 4 °C, followed by incubation with secondary antibodies for 30 min at room temperature. Next, DAB chromogenic solution (P0202; Beyotime, Jiangsu, China) was added to the slices, and the reaction was terminated by immersion in PBS. Finally, the sections were stained with hematoxylin, fixed with 95% alcohol, sealed with neutral resin, and observed and photographed under a light microscope. Anti-CXCR4 (Abcam, ab124824, 1:200) was used in this IHC procedure.

Flow cytometry

In order to detect changes in the numbers of EPCs in peripheral blood, CD34 and CD133 were used as markers of EPCs. Samples of orbital blood were collected into anticoagulant tubes and diluted with PBS. Next, reagent A and reagent D were added to form a gradient as described in instructions for use of peripheral blood monocyte cell separation liquid (P6700; Solarbio). Gradient centrifugation was performed to filter the mixture at room temperature, and the white monocyte layer was transferred into a 15 mL centrifuge tube and washed with PBS. Next, primary antibodies were added to the monocyte cells and incubated at 4 °C for 60 min at room temperature. After washing with PBS, the cells were incubated with secondary antibodies in the dark at 4 °C for 30 min. Finally, pre-cooled PBS was added to the monocytes and the cells were analyzed by flow cytometry (CytoFlex; Beckman Coulter). The antibodies used for flow cytometry studies were anti-CD133 (18470-1-AP, 0.2 µg/mL; Proteintech), anti-CD34 (60180-1-Ig, 0.2 µg/mL; Proteintech), goat anti-Rabbit IgG H&L (PE) (ab72465, 1:500; Abcam), and goat anti-mouse IgG H&L (FITC) (ab6785, 1:500; Abcam).

Real-time fluorescence quantitative PCR (qRT-PCR)

qRT-PCR was performed to detect the expression of SDF-1 and CXCR4 in the skin wound tissues of diabetic rats. Total RNA was extracted from skin wound tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using a PrimeScript™ RT reagent Kit (Takara, Shiga, Japan) as described in the manufacturer’s instructions. qRT-PCR was performed by using SYBRR Green Realtime Master Mix (Takara) on an ABI Real time PCR (7500) instrument. (with) The cycle conditions used for qRT-PCR were as follows: initial incubation for 3 mins at 95 °C, followed by 35 cycles of 5 s at 95 °C and 5 s at 60 °C. Relative levels of gene expression were calculated using the 2^−ΔΔCT method. The primers used for qRT-PCR are listed in Table 1.

Western blotting (WB)

A protein extraction kit (Beyotime, P0013J) was used to extract the total protein from rat skin ulcer tissues according to the manufacturer’s protocol. Prior to centrifugation at 12,000g for 15 min at 4 °C, TissueLyser beads were added to the centrifugation tubes to assist with protein cleavage. The upper layer of lipid was removed, and a BCA Protein Assay Kit (Beyotime, China) was using to determine the protein concentration in each extract. Western blotting was performed according to a standard WB protocol (Yukhananov, Chimento & Marlow, 2022). ImageJ software (v 1.8.0) was used to quantify protein levels, and GAPDH expression served as an internal standard. The primary antibodies used for WB were as follows: anti-CXCR4 (ab124824, 1:100; Abcam), anti-SDF1 (ab25117, 1:1,000; Abcam), and anti-GAPDH (ab9485, 1:2500; Abcam). The secondary antibody was goat anti-rabbit IgG H&L (HRP) (ab6721, 1:2000; Abcam).

Statistical analysis

All data were analyzed using IBM SPSS Statistics for Windows, Version 25 software (IBM Corp., Armonk, NY, USA) and graphs were drawn using GraphPad Prism 8.0 software. One-way ANOVA followed by Tukey’s post hoc test or the student’s t test was used to compare mean values among different groups. Quantitative data are expressed as a mean value ± SEM. A p-value < 0.05 was considered to be statistically significant.

TTT accelerated wound closure and promoted skin tissue recovery

Measurements of rat body weight taken before and after STZ injection revealed no significant change in body weight. However, there were significant differences in body weight among the three different groups (blank group vs. Fixator group vs. TTT group) at 9 days after the operation (Fig. 2B). Especially at 21 days following surgery, there was no discernible difference in body weight among the different treatment groups. In order to observe the healing of the ulcer after operation, the wounds were photographed on days 3, 6, 9, 12, and 15 after surgery. H&E staining was performed on days 8, 15, and 21 after surgery. When compared to the rats in the blank group, the wound healing areas of rats in the TTT group were the smallest, followed by the wound healing areas in the Fixator group, especially at 15 days after the operation (Fig. 2C). H&E staining was used to examine the regenerative capability of wounded skin (Fig. 2D). In the blank group, the cells of the wounded skin were discontinuous and broken, even at 21 days after the operation. After treatment with the TTT technique, the epidermal and dermal cells of wound skin were more closely and orderly arranged, and the thickness of the dermis was greater than that in the Fixator group. When taken together, these findings indicated that the TTT technique not only significantly accelerated the wound healing process, but also enhanced skin tissue regeneration and repair.

TTT increased the levels of EPCs to contribute to wind healing

The levels of EPCs in peripheral blood were detected by flow cytometry and using CD34 and CD133 as markers (Fig. 3A). Our data showed that at different time points after operation, the TTT technique significantly increased EPC levels when compared to levels in the blank group, and the differences became increasingly obvious over time. The numbers of CD34+CD133+ cells in the Fixator group were significantly greater than those in the blank group only at 21 days after surgery (Fig. 3B, p < 0.001). As the post-surgery time increased, the difference in EPC numbers between the TTT group and Fixator group steadily widened, indicating that TTT had a more rapid and pronounced effect.

TTT increased the numbers of EPCs and activated the SDF-1/CXCR4 signaling pathway

EPC levels were detected by the immunofluorescence of CD34 and CD133, and the deposition of SDF-1 and CXCR4 proteins was detected using immunofluorescence and immunohistochemistry; those results are summarized in Figs. 4–6. Our data showed that three indicators (CD34, CD133. and CXCR4) had increased expression levels in the TTT group, regardless of the time after surgery (Fig. 4A) and an IF quantitative analysis showed the same result (Figs. 4B–4D). The levels of fluorescence intensity in the fixator group were significantly higher than those in the blank group on days 15 and 21 after surgery. Similar trends in CD34, CD133, and SDF-1 expression are shown in Fig. 5A. Moreover, a quantitative examination of IF results was also conducted (Figs. 5B–5D). However, those results showed relatively higher fluorescence intensities occurring in the TTT group at 15 days after surgery rather than at 21 days after surgery.

Our results showed that CXCR4 was highly expressed in skin wounds in the TTT group and the levels of CXCR4-positive cells increased over time. On day 21 after surgery, high numbers of CXCR4 positive cells were found in both the fixator group and TTT group (Fig. 6A).

TTT treatment significantly increased the numbers of CXCR4-positive cells in the skin wounds of diabetic rats. Meanwhile, our statistical results were consistent with the phenomenon observed by IF (Fig. 6B).

TTT promoted SDF-1/CXCR4 expression

qRT-PCR and western blotting were used to measure SDF-1/CXCR4 expression in skin tissue, both quantitatively and qualitatively. Our data showed that TTT treatment increased the levels of CXCR4 expression in skin wound sites (Fig. 7A). Significant differences in SDF-1 mRNA expression between the TTT group and blank group were not found until 21 days after surgery (Fig. 7B). The effects of TTT surgery on SDF-1/CXCR4 protein expression in skin wound sites were consistent with those shown by mRNA analysis (Fig. 7C). High levels of SDF-1/CXCR4 proteins were expressed in the TTT group on days 9, 15, and 21 after surgery when compared to their expression in the blank group. On days 9 and 21 after surgery, the levels of CXCR4 and SDF-1 proteins were significantly increased in the fixator group when compared with those in the blank group (Figs. 7C–7E). Both the quantitative and qualitative experimental results showed that the levels of SDF-1/CXCR4 protein expression in the TTT group were significantly higher than those in the fixator or blank group. This indicated that the TTT technique contributed to skin wound healing by activating the SDF-1/CXCR4 signaling pathway. Overall, these findings showed that TTT promoted skin wound healing over time.