The Combination of Concentrated Growth Factor and Adipose-Derived Stem Cell Sheet Repairs Skull Defects in Rats

Abstract

Background:

The goal of this study was to create a biomaterial which combines concentrated growth factor (CGF) with an adipose-derived stem cell (ADSC) sheet to promote the repair of skull defects in rats.

METHODS:

We determined the optimal concentration of CGF extract by investigating the effects of different concentrations (0, 5%, 10%, and 20%) on the proliferation and differentiation of ADSCs. Then we created a complex combining CGF with an ADSC sheet, and tested the effects on bone repair in four experimental rat groups: (A) control; (B) ADSC sheet; (C) CGF particles; (D) combination of CGF + ADSCs. Eight weeks after the procedure, osteogenesis was assessed by micro-CT and hematoxylin and eosin staining.

RESULTS:

We found that the concentration of CGF extract that promoted optimal ADSC proliferation and differentiation in vitro was 20%. In turn, bone regeneration was promoted the most by the combination of CGF and ADSCs.

CONCLUSION:

In this study, we determined the optimal ratio of CGF and ADSCs to be used in a biomaterial for bone regeneration. The resulting CGF/ADSCs complex promotes maxillofacial bone defect repair in rats.

Introduction

Bone defects caused by infection, trauma or other reasons in the oral and maxillofacial regions are extremely common, but repair and regeneration can be quite difficult. Conventional repair methods like autologous bone grafting are commonly associated with secondary lesions and poor repair effectiveness.

With progress in cell biology and material science, novel approaches to bone tissue engineering and regeneration are becoming a reality.

Concentrated growth factor (CGF), an advanced, second generation platelet concentrate containing various growth factors and fibrin, is considered to work better than platelet rich-plasma (PRP) and platelet-rich fibrin (PRF) for promoting tissue regeneration [1, 2].

Adipose-derived mesenchymal stem cells (ADSCs), isolated from adipose tissue, exhibit multidirectional differentiation potential [3]. Compared with stem cells obtained from other sources, ADSCs offer many advantages, such as an abundant supply, simple preparation and strong regeneration [4]. Hence, they are becoming an important source of seed cells in tissue engineering [5, 6]. However, studies have found that simple cell implantation is associated with low cell survival [7].

To solve this problem, cell sheet technology is used in tissue engineering [8–10]. ADSCs sheets are three-dimensional (3D) structures containing large numbers of seed cells and abundant extracellular matrix. The extracellular matrix secreted by ADSCs acts as an autogenous scaffold to amplify the biological activities of ADSCs [11].

Stem cells could be crucial to promote bone regeneration. On the other hand, PRF/CGF can provide an autologous fibrin mesh scaffold for seed cell attachment with abundant growth factors. Therefore, many studies have explored the way in which cells attach and the optimal proportion of stem cells to PRF/CGF. For example, Zhao et al. [12] showed that platelet-rich fibrin granules enhanced the binding of periodontal ligament stem cells (PDLSCs). This approach promotes the release of factors by PDLSCs and in combination can significantly promote the healing of replanted teeth and periodontal tissues. Studies have also shown that medium containing CGF particles can promote the in vitro proliferation and bone differentiation of dog adipocyte stem cells [13].

According to previous studies, CGF can promote proliferation and bone differentiation of adipose stem cells in vitro [14]. Therefore, mechanical blending of ADSC sheets with CGF particles could potentiate the effects of ADSCs. However, the optimal ADSC and CGF ratio, and whether the complex of CGF and ADSCs can promote bone formation in vivo, is still unknown. Thus, our study aimed to identify the optimal ratio of ADSC to CGF and to demonstrate the effects of this complex on bone repair in vivo.

Materials and methods

Animals

Specific pathogen-free (SPF) Sprague-Dawley rats were purchased from the Hubei Center for Disease Control and Prevention. All animal procedures and treatments were performed in accordance with relevant ethical guidelines by the Hubei University of Medicine (No. 2019-105).

CGF preparation

Ten ml of blood from the abdominal aorta was collected in a negative pressure tube and centrifuged for 13 minutes in a Medifuge CGF apparatus (Silfradent, Santa Sofia FC, Italy) using a variable speed centrifugal program. The upper (serum) and bottom (red blood cells) layers were discarded and the middle gel-like layer (CGF) was repeatedly washed with phosphate buffer saline (PBS, Hyclone, Logan, UT, USA). The three-dimensional structure of CGF was analyzed by scanning electron microscopy (HITACHI, Tokyo, Japan) and its microstructure by hematoxylin and eosin (H&E) staining (Fig. 1).

Fig. 1.

Preparation and characterization of CGF. A, B Rat blood was separated into layers by centrifugation. The middle gel-like substance is CGF; C H&E staining of CGF. CGF is composed mainly of extracellular matrix with occasional cellular elements; D Ultrastructure of CGF observed under the scanning electron microscope. CGF showed a 3-dimensional network consisting predominantly of fibrin fibers

The CGF gel was frozen at − 80 °C for 1 h, and centrifuged at 4 °C after thawing (230 g, 10 min). Next, 5 ml α-MEM culture medium was added (Hyclone), samples were incubated at 37 °C for 24 h, and centrifuged (400 g, 5 min). Five ml of the supernatant were collected and filtered with a 0.22 µm filter to remove bacteria. This was the CGF extract [13, 15, 16].

Isolation and culture of ADSCs

Adipose tissue from the groin region of 2-week-old SD rats was removed under aseptic conditions. The tissue was cut into pieces, repeatedly washed with PBS and digested with 0.1% collagenase I enzyme (Gibco, Grand Island, NY, USA). After 50 min, α-MEM medium was added to terminate the reaction and cells were centrifuged at 1,000 r/min for 5 min. The primary ADSCs were transferred to a Petri dish, and cells were passaged following conventional procedures.

Determination of multidirectional differentiation potential of ADSCs

Third generation ADSCs were plated onto 6-well plates at a density of 1 × 10⁵ cells/well. After cell adherence, the culture media was replaced with osteogenic or lipogenic media (Cyagen US Inc., Santa Clara, CA, USA). After 21 days, the culture medium was removed and cells were fixed. Alizarin red staining (Solarbio, Beijing, China) was performed in the osteogenic differentiation group and oil red O staining (Solarbio) in the lipogenic differentiation group.

Cell proliferation assay

Third generation ADSCs in logarithmic growth phase were plated onto 96-well plates at a density of 2,000 cells/well and cultured in complete medium containing 0, 5%, 10% or 20% of CGF extract. Five replicates were set for each group. The cells were incubated at 37 °C with 5% CO₂ for 1, 3, 5, and 7 days. Next, 10 ul/well of CCK-8 solution was added (Beyotime, Shanghai, China) and cells were incubated for 1 h. Finally, the absorbance was measured at 450 nm.

ALP staining

Third generation ADSCs were plated onto 6-well plates at a density of 5 × 10⁴/ml. When confluence reached 80%, the cells were cultured in osteogenic differentiation medium containing 0, 5%, 10% or 20% of CGF extract. After 7 days, cells were stained for ALP (Beyotime).

RNA extraction, reverse transcription and quantitative real-time PCR (qRT-PCR)

Third generation ADSCs were plated onto 6-well plates at a density of 5 × 10⁴/ml. When confluence reached 80%, the cells were cultured in osteogenic differentiation medium containing 0, 5%, 10% or 20% of CGF extract. Total RNA was extracted from the cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was generated using the PrimeScript® RT reagent Kit (Takara, Dalian, China) and qRT-PCR was performed using SYBR Premix Ex Taq II (Takara). β-actin was used as the internal control for determining relative mRNA levels, which were calculated using the 2^-ΔΔCt method. The following primer sequences were used for qRT-PCR: ALP (forward) 5′-AGAACAGAACTGATGTGGAATATGAA-3′ and (reverse) 5′- CAGTGCGGTTCCAGACATAGT-3′; Runx2, (forward) 5′-TTCAAGGTTGTAGCCCTCGGAG-3′ and (reverse) 5′-TCAAAGTGAAACTCTTGCCTCGT-3′; β-actin, (forward) 5′-AGCCATGTACGTAGCC-ATCCA-3′ and (reverse) 5′-TCTCCGGAGTCCATCACAATG-3′. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China).

Protein extraction & Western blot

After inducing osteogenic differentiation of ADSCs for 7 days with medium containing 0, 5%, 10% or 20% of CGF extract, total proteins were extracted and their concentration measured by means of the BCA method (Beyotime, Shanghai, China).

Proteins were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membranes (Millipore, Beijing, China). The PVDF membranes were blocked with 5% non-fat milk, incubated overnight at 4 °C with ALP- or Runx2-specific antibodies (Cell Signaling Technology, Danvers, MA, USA) and then with secondary antibodies (Boster, Wuhan, China). Protein levels were normalized to β-actin (Boster).

Preparation of ADSC sheets

A total of 5×10⁵ third generation ADSCs in good condition were plated onto a 10 cm Petri dish. When confluence reached 80%, the cells were cultured in film-forming medium containing 50 ug/ml of ascorbic acid (Beyotime). After 30 days of incubation, a visible transparent white sheet appeared at the bottom of the dish (Fig. 2A), corresponding to the mature ADSC sheet [17]. Its 3D structure was analyzed by scanning electron microscopy (Fig. 2B).

Fig. 2.

Preparation and characterization of ADSC sheets. A ADSCs were induced to form a translucent cell sheet;B Ultrastructure of the ADSCs sheet observed under the scanning electron microscope. The cells interweave and adhere to each other

Preparation of ADSCs & CGF combination

The ADSC sheet was cut into 0.5 mm × 0.5 mm fragments, the CGF was cut into 0.5 mm × 0.5 mm × 0.5 mm particles, and then both were mechanically and evenly mixed at a 1:4 ratio.

Induction and repair of skull defects in SD rats

Thirty-two 8-week-old male SD rats were randomly divided into four groups (n = 8 per group) to receive different treatments: (A) control; (B) ADSC sheet; (C) CGF particles; (D) CGF & ADSCs sheet combination. After exposing the calvarium, two symmetrical defects were created using a 4-mm diameter trephine. The left calvarial defect was treated with different biomaterials, whereas the right defect was used as an internal (untreated) control. After 2 months, bone formation in the skull defect area was examined.

Microcomputed tomography (micro-CT) analysis

Eight weeks after the surgical procedure, animals were sacrificed by anesthetic overdose. The skulls were removed, fixed with 4% paraformaldehyde, and scanned with a micro-CT device (Scanco Medical, Bassersdorf, Switzerland) to generate 3D images. The bone defect area was quantified using Image J and the rate of osteogenesis was calculated.

Histological examination

The samples were fixed for 2-3 d; then, the tissues were decalcified in 18% EDTA for 2-3 weeks and embedded in paraffin. Histological sections were stained with H&E and photographed.

Statistical analysis

All data were analyzed with ANOVA by using SPSS 20.0. Results are expressed as the mean ± SD. Differences were considered statistically significant when p  <  0.05. *p < 0.05, **p < 0.01.

Results

Multidirectional differentiation of ADSCs

After inducing osteogenic differentiation of ADSCs for 21 days, Alizarin red staining showed numerous red mineralized particles under the microscope (Fig. 3A). After adipogenic differentiation of ADSCs, oil red O staining showed bright red lipid oil droplets in the cytoplasm (Fig. 3B).

Fig. 3.

Multidirectional differentiation of ADSCs. A Alizarin red staining (x40); B Oil red O staining (x40)

Cell proliferation measured by the CCK-8 assay

At each time point (except on the first day), cells exposed to the 20% CGF extract proliferated the most, and the absorbance values in the experimental groups were significantly higher than in the control group (P < 0.05) (Fig. 4).

Fig. 4.

Proliferation of ADSCs in the presence of different concentrations of CGF extract, measured with the CCK-8 assay. Each data point represents the mean ± SD, n = 3 per group. *p < 0.05, **p < 0.01 versus control

ALP staining

After 7 days of osteogenic differentiation, cells in the four experimental groups stained dark blue. However, the number of positive cells in the 20% CGF extract group was higher than in any other group (Fig. 5).

Fig. 5.

ALP staining (×40) of ADSCs after induction of osteogenesis: A without CGF; B in the presence of 5% CGF extract; C in the presence of 10% CGF extract; D in the presence of 20% CGF extract.

Levels of expression of bone-related mRNAs and proteins

Real-time quantitative PCR showed that ALP and Runx2 mRNA expression levels in the experimental groups were higher than in the control group. However, cells exposed to the 20% CGF extract showed the highest expression level (Fig. 6A).

Fig. 6.

Expression of osteogenic-related mRNAs and proteins. A ALP and Runx2 mRNA expression levels tested by qRT-PCR; B ALP and Runx2 protein levels tested by Western blot. Data are presented as mean ± SD, n = 3 per group. *Indicates significant differences between each groups, *p < 0.05, **p < 0.01

Western blot analysis showed that ALP and Runx2 protein expression was higher in the experimental groups than in the control group. In addition, the levels of expression correlated with the concentration of the CGF extract (Fig. 6B). Therefore, the Western blot results were consistent with the PCR results.

Micro-CT and histological assessment of bone regeneration in Vivo

The micro-CT analysis showed different degrees of new bone formation in the four experimental groups, with new bone growing mainly in the marginal area of the bone defect (Fig. 7). The rate of osteogenesis was as follows: (A) control (0.4 ± 2.85%), (B) ADSC sheet (16.22 ± 1.70%), (C) CGF particles (18.16 ± 2.56%), and ADSC sheet + CGF particles (27.98 ± 3.23%). Therefore, the ADSC sheet + CGF particle combination promoted the formation of new bone significantly better than the two biomaterials alone (Fig. 8).

Fig. 7.

3D images of the skull defects of rats 8 weeks after the surgical procedure, reconstructed by micro-CT: A control; B ADSC sheet particles; C CGF particles; D Complex of CGF & ADSCs. The left hole was used for the experiments, and the right hole was an internal (untreated) control

Fig. 8.

The bone defect area was quantified using Image J and the rate of osteogenesis was calculated. Data are presented as mean ± SD, n = 8 per group. *Indicates significant differences between each groups. *p < 0.05, **p < 0.01

Histological assessment of bone regeneration in Vivo

H&E staining showed that the bone defect area in the control and ADSC sheet groups contained mainly fibrotic connective tissue. In contrast, newly formed bone matrix was observed in both CGF particle and ADSC sheet + CGF particle groups, but with the latter group showing more evident bone regeneration (Fig. 9).

Fig. 9.

Histological assessment of bone formation in the defect 8 weeks after the procedure by H&E staining. Bone regeneration was most evident in the group treated with CGF & ADSCs: A control; B ADSCs sheet particles; C CGF particles; D Complex of CGF & ADSCs

Discussion

A defect or absence of the mandible caused by infection, trauma or tumor is always a problem in oral medicine. Due to secondary trauma caused by bone transplantation or immune rejection of artificial bone substitutes, traditional treatments many times fail to meet the needs of the patient.

Bone tissue regeneration is becoming a hot topic in stomatology. With stem cells and novel biomaterials, it is possible to develop new therapeutic alternatives. Many studies have shown the advantages of using ADSCs as seed cells in bone tissue engineering [18]. However, implantation of pure ADSCs is associated with a low survival rate, unsatisfactory osteogenic differentiation, and other problems [19].

The cell sheet technique induces seed cells to secrete large amounts of extracellular matrix, which forms a three-dimensional structure that promotes cell-cell signaling. The integrity of the extracellular matrix promotes the survival and function of cells [20]. This technique can overcome the problem of low cell activity.

CGF is a third generation extract prepared by centrifuging blood samples at alternating and controlled speeds and containing denser fibers and higher concentrations of growth factors [21]. These factors include TGF, PDGF, and BMPs, which can induce the migration, proliferation and differentiation of osteoblasts. Meanwhile, its gel-like consistency can slow down the diffusion of growth factors and prolong their effects. A stem cell sheet can enhance the survival of seed cells and CGF can supply factors needed for cell proliferation and differentiation, so it seems likely that the combination of the two materials can potentiate bone repair.

A study has confirmed that mechanical blending of PDLSC sheets and PRF particles enhances the effects of PRF on PDLSCs [12]. Due to structural similarities, this composite biomaterial should also be effective in the case of ADSCs and CGF. However, the optimal ratio of ADSCs to CGF is unknown, and whether it can promote bone formation in vivo is still unclear.

In this study, we obtained the CGF extract and identified the optimal concentration to promote the proliferation and differentiation of ADSCs, which was 20%.

Based on a conversion scale [12], we created a complex containing CGF and ADSCs with a volumetric ratio of 1 to 4, and tested its effectiveness in vivo with the bone repair experiment in rats.

In conclusion, our study demonstrates that CGF can significantly promote the proliferation and osteogenic differentiation of ADSCs in vitro, and that their combination shows good osteogenic effects in vivo. These results provide an experimental and theoretical basis for the investigation of CGF and ADSCs in bone regeneration research and for their potential clinical applications.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no financial conflicts of interest.

Ethical statement

The animal studies were performed after receiving approval from the Institutional Animal Care and Use Committee of the Hubei University of Medicine (No. 2019-105)