The Combination of Platelet Rich Plasma Gel, Human Umbilical Mesenchymal Stem Cells and Nanohydroxyapatite/polyamide 66 Promotes Angiogenesis and Bone Regeneration in Large Bone Defect

Abstract

Background: In the present study, a novel tissue engineering bone graft including platelet rich plasma gel (PRP gel), human umbilical mesenchymal stem cells (HUMSCs) and nanohydroxyapatite/polyamide 66 (nHA-PA66) was constructed. We explored whether the composite scaffolds could enhance the angiogenesis and bone repair capacity in rat femoral large bone defect (LBD). This study aimed to provide evidence for the clinical application of the composite scaffold in LBD treatment.

Methods: PRP was prepared, the platelets and growth factors were measured. HUMSCs were isolated and identified. the osteogenic capacity of PRP in vitro was measured. Then HUMSCs-PRP-gel/nHA-PA66 composite scaffolds were synthesized and observed. The proliferation and osteogenesis differentiation of HUMSCs on the composite scaffold was measured. The angiogenic capacity of PRP in vitro was measured by capillary-like tube formation assay. Finally, the angiogenesis and bone repair capacity of the composite scaffolds was measured in rat LBD.

Results: PRP contained high level of platelets and growth factors after activation, and promoted osteogenic and angiogenic differentiation in vitro. The HUMSCs-PRP-gel/nHA-PA66 composite scaffold was porosity and promoted the proliferation and osteogenesis differentiation of HUMSCs. At 12th weeks, more micro-vessels and new bone were formed around the composite scaffolds compared with other groups, the defect was almost repaired.

Conclusion: Our study for the first time identified that the combination of PRP gel, HUMSCs and nHA-PA66 scaffold could significantly promote angiogenesis and bone regeneration in rat LBD, which may have implications for its further application in clinical LBD treatment.

Keywords: Angiogenesis; Large bone defect; Nanohydroxyapatite/polyamide 66; Osteogenesis; Platelet rich plasma.

Fig. 1

Representative flow cytometry scatter plots, HUMSCs are a homogeneous population of cells and exhibit surface expression of standard cell surface markers. A Positive for CD 73 and CD 105. B Negative for CD 34 and CD 45

Fig. 2

PRP promotes osteogenic and angiogenic differentiation in vitro. The ALP-positive cells and calcium deposition was more prominent in OM + PRP group compared with OM group (40 ×). PRP significantly promoted the formation of capillary-like tubes in AM + PRP group. A ALP activity after 7 days of osteogenic induction. B Absorbance of alizarin red staining after 14 days of osteogenic induction. C, D The number and average diameters of formed tubes after 24 h of angiogenic induction. E Relative expression of COL1A1 and OCN mRNA after 14 days of osteogenic induction. F,G The protein expression of COL I and OCN after 14 days of osteogenic induction. (Data in mean ± SD, n = 5, ^*p < 0.5, ^**p < 0.01, ^***p < 0.001, OM = osteogenic medium, AM = angiogenic medium)

Fig. 2

PRP promotes osteogenic and angiogenic differentiation in vitro. The ALP-positive cells and calcium deposition was more prominent in OM + PRP group compared with OM group (40 ×). PRP significantly promoted the formation of capillary-like tubes in AM + PRP group. A ALP activity after 7 days of osteogenic induction. B Absorbance of alizarin red staining after 14 days of osteogenic induction. C, D The number and average diameters of formed tubes after 24 h of angiogenic induction. E Relative expression of COL1A1 and OCN mRNA after 14 days of osteogenic induction. F,G The protein expression of COL I and OCN after 14 days of osteogenic induction. (Data in mean ± SD, n = 5, ^*p < 0.5, ^**p < 0.01, ^***p < 0.001, OM = osteogenic medium, AM = angiogenic medium)

Fig. 3

The morphology of A nHA-PA66 scaffolds, B PRP-gel/nHAPA66, C HUMSCs/nHA-PA66 and D HUMSCs-PRP-gel/nHAPA66. The yellow areas represent the fibrin network formed by PRP gel. The green areas represent the stem cells adhered to the scaffolds. E The porosity of the scaffolds from the four groups. (Data in mean ± SD, n = 5)

Fig. 4

MTT assay for cell proliferation in vitro. The HUMSCs-PRP-gel/nHA-PA66 group demonstrated significantly higher absorbance values compared with the HUMSCs/nHA-PA66 group at day 3 and 7. (Data in mean ± SD, n = 5, ^*p < 0.05, ^**p < 0.01)

Fig. 5

ALP activity assay for cell osteogenic differentiation in vitro. The HUMSCs-PRP-gel/nHA-PA66 group demonstrated significantly higher ALP activity compared with the HUMSCs/nHA-PA66 group at day 3 and 7. (Data in mean ± SD, n = 5, ^*p < 0.05, ^**p < 0.01)

Fig. 6

Surgical procedure for the implantation of scaffolds in rat femoral LBD site. A The scaffold was cut into 5 × 5 × 5 mm³ before implantion. B Cylindrical LBD of 5 mm diameter and 5 mm depth was established in rat femur. C The scaffold could be accurately and stably filled into the femoral defect area

Fig. 7

HUMSCs-PRP-gel/nHA-PA66 significantly improved angiogenesis and morphological repair in bone defects at 6th week after surgery. A Relative area of new bone tissue in repair area. B Quantitative analysis of mean vessel density per mm². C Relative expression of PDGF-B, VEGFA and TGF-β1 mRNA in repair area. (3 randomized fields of view for each group. S: nHA-PA66 scaffold, NB: newly formed bone, F: fibrous and connective tissue, black arrows: microvessels, Scale bar = 100 μm. Data in mean ± SD, n = 5, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus Defect only group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 versus PRP-gel/nHA-PA66 group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus HUMSCs/nHA-PA66 group)

Fig. 8

A Two dimensional images by micro-CT of different groups at 12th week after surgery (dashed rectangles indicate bone defect sites). B Bone volume at 12th week after surgery. C Bone volume density at 12th week after surgery. D Bone mineral density of new bone tissues at 12th week after surgery. (3 randomized fields of view for each group, Data in mean ± SD, n = 5, ^*p < 0.05, ^***p < 0.001 versus Defect only group; ^&p < 0.05, ^&&&p < 0.001 vs. PRP-gel/nHA-PA66 group; ^#p < 0.05, ^##p < 0.01 versus HUMSCs/nHA-PA66 group)

Fig. 8

A Two dimensional images by micro-CT of different groups at 12th week after surgery (dashed rectangles indicate bone defect sites). B Bone volume at 12th week after surgery. C Bone volume density at 12th week after surgery. D Bone mineral density of new bone tissues at 12th week after surgery. (3 randomized fields of view for each group, Data in mean ± SD, n = 5, ^*p < 0.05, ^***p < 0.001 versus Defect only group; ^&p < 0.05, ^&&&p < 0.001 vs. PRP-gel/nHA-PA66 group; ^#p < 0.05, ^##p < 0.01 versus HUMSCs/nHA-PA66 group)