A comparison of tissue engineering based repair of calvarial defects using adipose stem cells from normal and osteoporotic rats

Abstract

Repairing large bone defects presents a significant challenge, especially in those people who have a limited regenerative capacity such as in osteoporotic (OP) patients. The aim of this study was to compare adipose stem cells (ASCs) from both normal (NORM) and ovariectomized (OVX) rats in osteogenic potential using both in vitro and in vivo models. After successful establishment of a rat OP model, we found that ASCs from OVX rats exhibited a comparable proliferation capacity to those from NORM rats but had significantly higher adipogenic and relatively lower osteogenic potential. Thirty-two weeks post-implantation with poly(lactic-co-glycolic acid) (PLGA) alone or PLGA seeded with osteogenic-induced ASCs for critical-size calvarial defects, the data from Herovici's collagen staining and micro-computed tomography suggested that the implantation of ASC-PLGA constructs exhibited a higher bone volume density compared to the PLGA alone group, especially in the NORM rat group. Intriguingly, the defects from OVX rats exhibited a higher bone volume density compared to NORM rats, especially for implantation of the PLGA alone group. Our results indicated that ASC based tissue constructs are more beneficial for the repair of calvarial defects in NORM rats while implantation of PLGA scaffold contributed to defect regeneration in OVX rats.

Keywords: Adipose stem cells; Calvarial defect; Osteogenesis; Osteoporosis; Poly(lactic-co-glycolic acid).

Fig. 1

Establishment of an OP model using OVX rats. Six-month-old SD rats underwent either bilateral ovariectomy (OVX) or sham surgery (NORM). Four months post-operatively, the rats from both OVX and NORM groups were compared in weight (A), % fat in spine region (B), and spine bone mineral density (BMD) (C). Data are shown as average ± standard deviation for n=5. *p < 0.05 indicated a statistically significant difference.

Fig. 2

Evaluation of in vitro proliferative potential of ASCs from both OVX and NORM rats. Passage 2 ASCs were expanded at 3000 cells/cm² for one passage. Expanded cells were collected at days 0, 2, 4, 6, and 8 for the measurement of DNA content using the Quant-iT™PicoGreen^® dsDNA assay kit with a CytoFluor^® Series 4000 (A). Expanded cells at days 4 and 8 were also measured for proliferation index using a BD FACS Calibur™flow cytometry (B).

Fig. 3

Evaluation of in vitro osteogenic potential of ASCs from both OVX and NORM rats. After a 21-day-osteogenic induction, ASCs were evaluated for osteogenic differentiation using ALP staining (A) and activity (B) as well as ARS staining (C) and osteogenic marker genes including BGLAP, RUNX2, and SPP1 (D). Data are shown as average ± standard deviation for n=4. *p<0.05 indicated a statistically significant difference.

Fig. 4

Evaluation of in vitro adipogenic potential of ASCs from both OVX and NORM rats. After a 21-day-adipogenic induction, ASCs were evaluated for adipogenic differentiation using ORO staining for lipid-filled droplets (A) and chemical quantitation of staining (B) as well as real-time PCR for adipogenic marker genes including PPAR, CEBP, and LPL (C). GM: Growth Medium; AM: Adipogenic Medium. Data are shown as average ± standard deviation for n=4. *p<0.05 indicated a statistically significant difference.

Fig. 5

ASC morphology and extracellular matrix as well as PLGA mesh. SEM was used to characterize morphological properties of PLGA mesh and ASCs and secreted matrix at 3 days (A/B) and 14 days (C/D) post-cell seeding and osteogenic induction. Scale for low magnification is 1 mm (A/C); scale for high magnification is 300 µm (B/D).

Fig. 6

Calvarial defect model and early-stage bone regeneration evaluation. Calvarial defects of 5 mm created bilaterally in both OVX and NORM rats were either left empty (A) or implanted with PLGA plus osteogenically induced ASCs or PLGA alone (B). Six weeks post-operatively, the defects were evaluated for bone formation using DEXA (C). Defect BMD ratio was defined as (R1 or R2)/[(R3+R4+R5+R6+R7)/5]. Data are shown as the average for n=6 defects in the NORM rat group (D) and n=4 defects in the OVX rat group (E).

Fig. 7

Evaluation of late-stage bone regeneration of calvarial defects in SD rats using histological analysis and µCT. Calvarial defects from NORM (A) and OVX rats (B) were implanted with PLGA plus osteogenically induced ASCs or PLGA alone for 32 weeks with untreated groups as empty controls (C and D, respectively). Representative images of calvarial bone sections were with Herovici’s collagen staining (10× original magnification). Arrows point to the defect margins. In the defect area, blue indicates new collagen while red indicates old collagen.

Fig. 8

Quantitative µCT analysis of 32-week bone formation in vivo using the ratio of bone volume (BV) to total volume (TV). The implantation with PLGA plus osteogenically induced ASCs and PLGA alone was evaluated for bone regeneration in NORM rats and OVX rats. Data are shown as average ± standard deviation for n=8 in the NORM rat group and n=7 in the OVX rat group. *p<0.05 indicated a statistically significant difference.