Bone regeneration by BMP-2 enhanced adipose stem cells loading on alginate gel

Histochem Cell Biol (2008) 129:203–210

ORIGINAL PAPER

Bone regeneration by BMP-2 enhanced adipose stem cells loading on alginate gel

Yunfeng Lin · Wei Tang · Ling Wu · Wei Jing · Xiaoyu Li · Yao Wu · Lei Liu · Jie Long · Weidong Tian

Accepted: 19 October 2007 / Published online: 3 November 2007© Springer-Verlag 2007

Abstract Adipose stem cells (ASCs) have the potential todiVerentiate into a variety of cell lineages both in vitro andin vivo. In this study, ASCs were harvested from normalSprague–Dawley (SD) rats and transfected by BMP-2 genebefore they were loaded on alginate. The ability of boneregeneration was determined in rat critical-size cranialdefects. An 8-mm diameter defect was created in the calva-rias of 36 rats; these rats were divided into three groups. Inexperimental group, the defects were Wlled with alginate gelcombined with BMP-2 transfected ASCs; in negative con-trol group, the defects were Wlled with alginate gel mixedwith normal ASCs; in blank controls, the defects were Wlledwith cell-free alginate gel. Four rats of each group werekilled and the cranial defect sites were observed at 4, 8 and16 weeks after surgery. There was complete repair of cra-nial defects in experimental group using the alginate gelloading BMP-2 transfected ASC, but only partial repair innegative controls and in the blank control. The engineeringapproach combining BMP-2 enhanced ASCs with alginategel can therefore stimulate bone regeneration and repair forthe large size bone defects.

Keywords Adipose stem cells · BMP-2 · Transfection · Alginate gel · Bone regeneration

Introduction

Large bone defects usually occur as a result of trauma,tumor or severe inXammation. Damaged sites have limitedpotential to heal, and hence large lesions never heal sponta-neously in orthopedic patients (Pelissier et al. 2003; Khanet al. 2005). Autogenously osteochondral grafts require aninvasive operation and are limited with the amount of avail-able tissue, donor site morbidity. Allografts are limited inusage owing to immunologic rejection, possible transmis-sion of infectious diseases, and donor shortage. Currently,the employment of tissue engineering substitutes hasenabled the creation of large functional bone tissues torestore the bone defect (Cancedda et al. 2007).

The seeding cell is the key factor of bone tissueengineering (Langer and Vacanti 1993). Based on previousworks and our own studies, adipose tissue is derived fromthe embryonic mesenchyme and contains multipotentialadipose stem cells (ASCs) (Jaiswal et al. 1997; Lin et al.2006a). Compared with BMSCs, ASCs are easier to obtain,have relatively lower donor site morbidity, grow fast, andare available in large number of stem cells at harvest from asmall volume of adipose tissue (Lin et al. 2006b) Thus,they are thought to be a promising alternative for BMSCsand an abundant source of cells for cell-based musculoskel-etal tissue engineering applications (Jaiswal et al. 2000;Gronthos et al. 2003).

With increasingly understanding of the molecularmechanisms during bone regeneration, now it is possible toaccelerate the bone healing and regeneration by genetransfection (Zhang et al. 2006; Wu et al. 2007). In theprocess of bone healing, bone morphogenetic protein-2(BMP-2) is a pleiotropic regulator, governing the key stepsin bone induction cascade such as chemotaxis, mitosis, anddiVerentiation of mesenchymal stem cells (Mie et al. 2000;

Y. Lin · W. Tang · L. Wu · W. Jing · X. Li · Y. Wu · L. Liu · J. Long · W. Tian (&)Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu 610041, Chinae-mail: [email protected]

Y. LinState Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China

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Wei et al. 2006). Although there have been some reportsdescribing the eVectiveness of BMP-2 for the osteogenesisin BMSCs and ASCs, it is unclear whether BMP-2enhanced ASCs can heal the large bone defects (Cowanet al. 2005; Dragoo et al. 2005; Saito et al. 2005; Knippen-berg et al. 2006; Li et al. 2007).

The alginate gel is one of the most extensively appliedbiomaterials in bone tissue engineering. In the presence ofcalcium ions, the semisolid gel can be formed with cross-linking of alginate chains under mild conditions (Cai et al.2007). It has been reported to have a high porosity, idealporous structure, biodegradable, biocompatibility, and highaYnity to water (Lee et al. 2007), which is necessary forengineering scaVolds. When cells are encapsulated intoalginate gel, interconnected pore can promote cell attach-ment, proliferation, and diVerentiation, and provides path-ways for nutrients and proteins diVusion (Ohta et al. 2004;Lin et al. 2005a). So this study attempted to deWne thepotential of BMP-2 enhanced ASCs loading on alginate gelto heal the critical-size cranial defects.

Materials and methods

Isolation and culture of ASCs

Thirty-six SD rats (West China Experimental Animal Cen-ter) aged 100 days and weighing about 250 g were used forthis study and their use was in line with the InternationalGuiding Principles for Animal Research. All surgical pro-cedures were done under approved anesthetic methods withNembutal at 35 mg/kg. The inguinal fat pads were har-vested and extensively washed with sterile phosphate-buVered saline (PBS). They were then excised and incu-bated with 0.075% type I collagenase (Sigma–Aldrich, StLouis, MO) in PBS for 60 min at 37°C with vigorous agita-tion. After neutralization of the collagenase, cells releasedfrom adipose specimens were Wltered and collected by cen-trifugation at 1,200g for 10 min. And then, the pellet wassuspended, washed three times with medium, and seededon the plastic tissue culture dishes in control medium con-taining �-MEM, 10% fetal bovine serum (FBS) (Lin et al.2005b; Lin et al. 2006a). ASCs were maintained in ahumidiWed atmosphere of 5% CO2 at 37°C and passagedthree times prior to gene transfection.

Gene transfer in vitro

ASCs from 12 rats (experimental group) were culturedin 6-well plates (2 £ 105 cells/well) were treated withDNA/LipoGen complexes at subconXuence. The plasmidpcDNA3.1-BMP-2, containing human BMP-2 cDNA (gen-erously provided by Dr L. J. Yang, Department of Oral

Pathology, Fourth Military Medical University, Xi’an,China), was mixed with LipoGen at a ratio of 1.5 �g/6 �l.After 72 h culture, the medium was replaced by controlmedium.

ImmunoXuoencent analysis of BMP-2 enhanced ASCs

Monolayer slides of BMP-2 transfected ASCs were pre-pared for immunoXuoencent analysis and Wxed in 4%buVered paraformaldehyde. Fixed glass slides were incu-bated with 3% hydrogen peroxide in methanol for 30 min.After washed with PBS, they were blocked in 1% bovineserum albumin and 1.5% normal goat serum at room tem-perature for 30 min. Slides were then incubated overnightat 4°C with rabbit anti-mouse polyclonal antibodies againstOCN (Santa Cruz, USA), and goat anti-mouse polyclonalantibodies against Runx2 (Santa Cruz, USA). Sequentially,slides were then incubated with secondary Xuorescent anti-bodies, including red Xuorescent goat anti-rabbit IgG(Invitrogen, USA) and green Xuorescent swine anti-goatIgG (SBA, USA). After rinsing in distilled water and cov-ered with slips, the slides were observed under Xuorescentmicroscopy. The proportion of positive staining cells wasquantitatively analyzed with a color image analysis system(Image-Pro Plus 5.0). The positive ratio, deWned as thenumber of cells with positive staining divided by the totalnumber of cells counted and expressed as a percentage, wascalculated.

Preparation of ASCs cells and alginate gel mixture

Allowed for 5 days recovery after the gene transfection,ASCs cells were suspended in 1.2% low-viscosity alginatein 0.15 mol/L NaCl at a density of 1 £ 107 ml¡1, and thenthe 102 mM CaCl2 solution were added with gentle agita-tion. After 15 min of polymerization at room temperature,the mixture was washed three times with normal saline,then three more times with the control medium (Lin et al.2005a). One-milliliter alginate gel mixed with BMP-2transfected and non-transfected ASCs cells or the acellularcontrol gel was prepared to heal the cranial defect.

In situ repair of critical-size cranial defects

All the 36 animals were anesthetized by Nembutal at35 mg/kg. The cranial bone surface of the region wasexposed widely and an 8 mm marker in nominal diameterwas measured using a surgical trephine. Removingperiosteum and cranial bone ensures adequate removal ofall tissue with osteoinductive and osteoconductivepotential. Then the defects were Wlled with alginate gelloading BMP-2 enhanced ASCs in 12 rats as experimentalgroup; 12 rats as negative controls, the defects were Wlled

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with alginate gel loading normal ASCs; while in 12 rats asblank controls the defects were Wlled only with acellularalginate gel. Four rats of each group were sacriWced andthe cranial defect sites were harvested to furtherexamination at 4, 8 and 16 weeks post-surgery (Lin et al.2007).

Histologic analysis

Retrieved samples were divided into three pieces. One wasstored at liquid nitrogen and the other two were Wxed in 4%buVered paraformaldehyde for 1 week. Of the two Wxedspecimens, one was cut into serial 50-�m-thick sectionswith a hard-tissue microtome (Leica, Cambridge, UK). Theother Wxed specimen was treated in decalciWed solution(50% formic acid + 20% sodium citrate, 1:1) for 4 weeksand then underwent procedures for dehydration,embedding, and incising. Hematoxylin and eosin stainingand Masson’s trichrome method were used to evaluate thebone regeneration at experimental sites.

RNA isolation and reverse transcription-polymerase chain reaction

Total RNA was extracted from all the specimens using theTRIzol Reagent (Life Technologies, Rockville, MD)according to the protocol. About 1�g of total RNA wasreversed transcribed by murine leukemia virus reversetranscriptase (TaKaRa, Japan) and PCR ampliWcation oftarget message RNA was performed by TaKaRa PCR kit(TaKaRa, Japan). PCR oligonucleotide primers andannealing temperature were listed in Table 1. The productswere electrophoresed on 1.5% agarose gels, stained withethidium bromide and visualized with Quantity Onesoftware (BIO-RAD).

Western blot analysis

Harvested tissue (100 mg) was homogenized and lysed in500 �l of lysis buVer. Five million cells from variantconditions were washed twice with PBS and also lysed inthe above buVer. After 14,000g centrifugation for 15 min,the supernatant was collected, and protein concentrationswere determined by the BCA assay (Pierce). Equal amountsof protein extracts were fractionated by 10% sodiumdodecyl sulfate-polyacrylamide gels, and electrophoreti-cally transferred to a nitrocellulose membrane (Bio-Rad).These nitrocelluloses were incubated with mousemonoclonal antibodies against RUNX2 and OCN (SantaCruz), rabbit polyclonal antibody to BMP2 (Abcam) andhousekeeping gene �-actin (Sigma). The bands were scannedand evaluated by Quantity One software (BIO-RAD).

Statistical analysis

Three or more independent sets of the experiments wereperformed, and each experiment was run at least threetimes. Data were expressed as means § SD and analyzedby a paired analysis of variance. P values were described inWgures and P < 0.05 was considered statistically signiWcant.

Results

Morphological features of primary cultures

Approximately 5 £ 105 nucleated cells were yielded fromthe inguinal fat pads of each mouse. Most of the cells thatattached to the culture dish surface exhibited a Wbroblast-like spindle shape at Wrst. They proliferated quickly incontrol medium to form colonies that grew and merged to a

Table 1 SpeciWc primers for PCR ampliWcation listed with expected fragments size and optimal annealing temperature

SpeciWc primers were designed following the cDNA sequences of each gene in GenBank. The upstream primer is shown above the downstreamprimer in all cases. The expected fragments size upon ampliWcation is given with optimal annealing temperature

Gene Primers Annealing temperature (°C)

Fragment (bp)

GenBank no.

OCN 5�-AACATAGTGTCGTCGTTTCTTTCTG-3� 5�-TATCAAACCAGTATGGCTTAAAGACC-3�

60 360 NM_031368

OPN 5�-ACTTGGTGGTGATCTAGTGGTGC-3� 5�-TCCAAGCAATTCCAATGAAAGCCAT-3�

60 337 NM_009263

RUNX2 F: 5�-GTGCCCAGGCGTATTTCA-3� R: 5�-CAGCGTCAACACCATCATTC-3�

56 487 NM_009820

BMP-2 5�-ACGTCCTCAGCGAGTTTGAG-3� 5�-CACCTGGCTTCTCCTCTAAG-3�

60 570 NM_017178

�-actin 5�-ACTCTTCCAGCCTTCCTTCC-3� 5�-ACTCGTCATACTCCTGCTTGC-3�

55 313 BC013835

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uniform conXuent cell monolayer after 2 weeks. Thecultures were passaged three times for expansion (Fig. 1a).

Cell transfection and examination of osteogenesis related gene and protein expression

Optimization experiments were performed with the plasmidpcDNA3.1-BMP-2, carrying a human BMP-2 gene, whilethe optimal liposome/DNA ratio for liposome-mediatedtransfection was 6 �l/1.5 �g to 2 £ 105 cells; the cellsshrink obviously after the transfection, but recovered soon(Fig. 1b). The expression of OCN (Fig. 1c) and RUNX2(Fig. 1d) in ASCs was detected by immunocytochemistry7 days after gene transfection, the positive ratio is nearly30–40%. In the control group, the immunocytochemistry ofthe non-transfected cells were negative. The RT-PCR(Fig. 4) and Western blotting (Fig. 5) analysis of the trans-fected ASCs were also performed to detect the expressionof bone related genes and protein.

In situ bone regeneration

No clinical sign of inXammation was observed in any ratsafter operations. Gross bone formation was traced during16 weeks in all cranial defects. At 4 weeks in experimentalgroup, the initial resorption of alginate scaVolds was clearand the newly formed bone extended. The consolidatedwhitish bone was found within the defect margin in

experimental group from 8 weeks on. At 8 weeks, therewas a great extent of reduction of cranial defects and theyaccomplished complete osseous healing at 16 weeks whenthe experiment Wnished. The bone formation in the negativecontrol groups was only presented disorderly along theperiphery of the defects and the central domain showedWbrous healing. There was few new bones formation at theblank control group.

In experimental groups, aggregation of cartilaginous andosteoid tissue were observed, the HE staining (Fig. 2a) andMasson’s trichrome staining (Fig. 2b) prove that in somecases weak osteogenesis was noted in the epidural region ofthe border of the defect at 8 weeks. After 16 weeks of treat-ment, the continued formations of new bone throughout thedefects were observed. The HE staining (Fig. 2c) and Mas-son’s trichrome staining (Fig. 2d) showed that the newbone network grew in nice continuity with the surroundinghost bone and spread homogeneously inside the wholedefect volume. As for the negative control groups, theirborder of the defects showed a small amount of new bonetissue and their opening was mainly Wlled with dense con-nective tissue rich in Wbroblasts and blood vessels at8 weeks. After 16 weeks, the HE staining (Fig. 3a) andMasson’s trichrome staining (Fig. 3b) showed that the boneislets formed by interstitial osteogenesis were observed inWbrous connective tissue. In the blank control group, thealginate gel was absorbed obviously at 4 weeks. The HEstaining (Fig. 3c) and Masson’s trichrome staining (Fig. 3d)

Fig. 1 The primary ASCs were the cells Wbroblast-like spindle shape (a). After the BMP-2 gene transfection, the ASCs shrink obviously, but recovered soon (b). After 7 days for recovery, the expression of OCN (c) and RUNX2 (d) in ASCs was detected by immunocyto-chemistry

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proved that there was no obvious new bone formation evenat 16 weeks, only dense connective tissue was found.

The radiographic image analyses of the defect in thediVerent groups were performed to evaluate the bone

healing eYciency. Just after the surgery of cranial defects,the mean area of the defects was 20 mm2. In the 8-weekexperimental groups, the mean area of the defects was10.27 § 0.45, respectively, with signiWcant decreases

Fig. 2 In experimental groups, He staining (a) and Masson’s trichrome method (b) proved the aggregation of cartilaginous and osteoid tissue, and in some cases weak osteogenesis was noted in the epidural region of the border of the defect at 8 weeks. After 16 weeks of treatment, the continued formations of new bone throughout the defects were observed. He staining (c) and Masson’s trichrome method (d) showed that the new bone network grew in perfect continuity with the surrounding host bone and spread homoge-neously inside the whole defect volume. (Bar = 50 �m)

Fig. 3 In the negative control group, HE staining (a) and Mas-son’s trichrome staining (b) show that bone islets formed by interstitial osteogenesis were ob-served in Wbrous connective tis-sue after 16 weeks. In the blank control group, the alginate gel was absorbed obviously at 4 weeks. HE staining (c) and Masson’s trichrome staining (d) proved that there was no obvious new bone formation even at 16 weeks, only dense connective tissue were found. (Bar = 50 �m)

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compared to the initial defects (P < 0.05). To the end of theexperiment, the defects healed completely with new bone.In the negative control group, till 8 weeks after surgery, thedefects showed no obvious reduction in size in comparisonwith the initial defects. At 16 weeks, few new bonesformed at the border of the defect. However, their meandefect area (14.89 § 0.49 mm2) was still larger thanexperimental ones (P < 0.05). In the blank control group,few new bones were formed around the defect at 16 weeks;the average area of defects was 16.68 § 0.55 mm2.

RT-PCR and western blotting analysis

The RT-PCR analysis of OPN, OCN, RUNX2 and BMP2demonstrated that there was signiWcant diVerence inexpression between experimental and control groups.

Continued high expression of OPN, OCN, RUNX2 andBMP2 was observed throughout the progression of theexperiment group both in vitro and in vivo. In negativecontrol groups, these genes were not observed in vitro and8 weeks in vivo, only at the 16 weeks after surgery, weakexpression of these genes was observed; in the blankcontrol group, these genes were not detected at 8 and16 weeks (Fig. 4).

The western blotting analysis of Runx2, OCN andBMP2 results was similar to the RT-PCR results. In theexperiment group, these proteins were observed in themonolayer cells after BMP-2 transfection. The in vivoanalysis still proved these protein expressions in 8th and16th week with an inclination. But these proteins were notobserved in the negative and blank control groups(Fig. 5).

Fig. 4 The RT-PCR analysis of OPN, OCN, RUNX2 and BMP-2demonstrated that there was signiWcant diVerence in expressionbetween experimental and control groups. Continued high expressionof OPN, OCN, RUNX2 and BMP-2 was observed throughout theprogression of the experiment group both in vitro and in vivo. In

negative control groups, these genes were not observed in vitro and8 weeks in vivo, only at the 16 weeks after surgery, weak expressionof these genes was observed; in the blank control group, these geneswere not detected at 8 and 16 weeks

Fig. 5 The western blotting analysis of Runx2, OCN and BMP-2 results was similar to the RT-PCR results. In the experiment group, these proteins were observed in the monolayer cells after BMP-2 transfection. The in vivo analysis still proved these protein expressions in 8th and 16th week with an inclination. But these proteins were not observed in the negative and blank control groups

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Discussion

New techniques involving implantation of cells and tissue-engineered constructs are being developed to improvemusculoskeletal tissue repair (Cancedda et al. 2007; Parket al. 2007). From a tissue regeneration point of view,ASCs can be applied in broad Welds clinically. If allied withother biological means, for example, utilizing electropora-tion, chemical reagents including calcium phosphate pre-cipitation and lipofection to transfected plasmids or viralvectors carrying genes encoding growth factors into ASCs,the engineered tissue can be formed even more eYciently(Dragoo et al. 2003; Li et al. 2007). It may lead to a betteroutcome that genetically modiWed ASCs seeded inconstructs are implanted in the defects immediately, whichfacilitates retention of high level expression locally andadaptation to the adjacent tissues.

A critical size defect is one that will not regeneratespontaneously during the term of an experiment, in thisresearch, which was a defect of 8 mm diameter on thecalvaria of a rat. After removal of the periosteum, apotential osteogenic inducer, this size defect had beenshown to maintain its size throughout a 4-month period(Cowan et al. 2004; Lin et al. 2007). The alginate implantsWlled with BMP-2 enhanced ASCs produced complete newbone repair within a relative short time after implantation atbony sites, similar to the BMSCs in previous research(Ohta et al. 2004; Lin et al. 2005a; Cai et al. 2007). Theresult in this paper indicated that alginate gel with BMP-2enhanced ASCs was necessary for critical size defect repairbecause intrinsic bone repair process was not powerfulenough, as shown in the control site. In the present study,the BMP-2 enhanced ASCs were well distributed inalginate system after mixture and produced suYcient ECMin the implants to form chondroid or osteoid aggregates(Saito et al. 2004). Engineering materials for constructs aretypically designed to provide a space for inWltrating cells toattach, proliferate, and produce new ECM (Pelissier et al.2003; Kang et al. 2007). Previous studies have clariWed thatalginate gel is injectable, which can transplant cells in aminimally invasive manner, and is suitable for lesions withirregular shapes.

To clarify the molecular events leading to the formationof new bone, we investigated expression of biochemicalmarkers using RT-PCR and western blotting along thecourse of BMP-2 enhanced ASCs diVerentiation. Thepositive RT-PCR bands of OCN, OPN, RUNX2 and BMP-2 conWrmed the osteogenesis in experimental groups. OPNis an early osteogenic marker and characterized in thematrix maturation and bone remodeling phase (Balint et al.2003; Porter et al. 2006). OCN is a late bone marker onlysecreted by osteoblasts and it also signals terminal osteoblastdiVerentiation (Mizuno et al. 2003). Its expression inside the

cranial defects made sure that osteogenesis and maturationof BMP-2 enhanced ASCs occurred.

Based on the results of complete repair of rat critical-sizecranial defects in the experimental group, load-bearingalginate with BMP-2 enhanced ASCs can therefore appliedin the engineering approach for further clinical usage.

Acknowledgments This work was supported by Opening Fundingof State Key Laboratory of Oral Diseases (Sichuan University)(SKLODKF200701), National Natural Science Foundation of China(30200318, 30560164), China Ministry of Science and Technologyunder Contract Preliminary Project (2002CCC00700, 2006CB708505), Science Foundation for The Excellent Youth Scholars ofMinistry of Education of China (2003682).

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