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Macroporous biodegradable natural/synthetic hybrid scaffolds as smallintestine submucosa impregnatedpoly(D, L-lactide-co-glycolide) fortissue-engineered boneSang Jin Lee , Il Woo Lee , Young Moo Lee , Hai Bang Lee &Gilson Khang

To cite this article: Sang Jin Lee , Il Woo Lee , Young Moo Lee , Hai Bang Lee & Gilson Khang(2004) Macroporous biodegradable natural/synthetic hybrid scaffolds as small intestinesubmucosa impregnated poly(D, L-lactide-co-glycolide) for tissue-engineered bone, Journal ofBiomaterials Science, Polymer Edition, 15:8, 1003-1017, DOI: 10.1163/1568562041526487

To link to this article: http://dx.doi.org/10.1163/1568562041526487

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J. Biomater. Sci. Polymer Edn, Vol. 15, No. 8, pp. 1003–1017 (2004) VSP 2004.Also available online - www.vsppub.com

Macroporous biodegradable natural/synthetic hybridscaffolds as small intestine submucosa impregnatedpoly(D,L-lactide-co-glycolide) for tissue-engineered bone

SANG JIN LEE 1,3, IL WOO LEE 2, YOUNG MOO LEE 3, HAI BANG LEE 1

and GILSON KHANG 4,∗1 Biomaterials Laboratory, Korea Research Institute of Chemical Technology, P. O. Box 107,

Yuseong, Daejeon 305-600, South Korea2 Department of Neurosurgery, Catholic University, Medical School, 520-2, Deaheung-dong,

Joong-gu, Daejeon 301-723, South Korea3 School of Chemical Engineering, Hanyang University, 17, Haengdang-dong, Seongdong-gu,

Seoul 133-791, South Korea4 Department of Polymer Science and Technology, Chonbuk National University, 664-14,

Dukjin-dong 1 Ga, Dukjin-gu, Jeonju 561-756, South Korea

Received 10 September 2003; accepted 4 February 2004

Abstract—Poly(D,L-lactide-co-glycolide) (PLGA), a biodegradable synthetic polymer, is widelyused in a variety of tissue-engineered applications, including drug-delivery systems. However, thePLGA scaffolds, macroporous and three-dimensional structure, are difficult to cell attachment andin-growth due to surface hydrophobicity. In order to introduce in new bioactive functionality fromporcine small intestine submucosa (SIS) as natural source for PLGA, we fabricated SIS-powder-impregnated PLGA (SIS/PLGA) hybrid scaffolds. Fabrication parameters, including ratios of SIS,PLGA and salt, were optimized to produce the desired macroporous foam. The scaffolds had arelatively homogeneous pore structure, good interconnected pores from the surface to core region andshowed an average pore size in the range 69.23–105.82 µm and over 90% porosity. The SIS/PLGAscaffolds degraded with a rate depending on the contents of the SIS. After the fabrication of theSIS/PLGA hybrid scaffolds the wettability of the scaffold was greatly enhanced, resulting in uniformcell seeding and distribution. So, it was observed that BMSC attachment to the SIS/PLGA scaffoldsincreased gradually with increasing SIS contents. Scaffolds of PLGA alone and SIS/PLGA wereimplanted subcutaneously under dorsal skin of athymic nude mouse to observe the osteoconductivity.It was found from the result that the effects of the SIS/PLGA scaffolds on bone formation are strongerthan control PLGA scaffolds. In summary, the SIS/PLGA scaffolds have osteoconductive effects toallow remodeling and replacement by osseous tissue.Key words: Poly(D,L-lactide-co-glycolide); small intestine submucosa; osteoconductivity; tissueengineering.

∗To whom correspondence should be addressed. Tel.: (82-63) 270-2336. Fax: (82-63) 270-2341.E-mail: gskhang@chonbuk.ac.kr

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INTRODUCTION

The concept of tissue engineering represents a construction containing selective vi-able cells, biological signaling molecules and biodegradable scaffolds in a syntheticor biologic matrix that can be implanted in patients to promote regeneration of dam-aged tissues [1–5]. Recently, this is done by providing natural and synthetic porousscaffolds, which mimics the body’s own extracellular matrix (ECM), onto whichcells attach, multiply, migrate and function. Therefore, to reconstruct new tissues,scaffolds, which cells are attached and cultured resulting in the implantation at thedesired site of the functioning tissue must be needed.

Tissue engineered scaffolds must be designed to satisfy various requirements.High porosity is needed for cell in-growth (>90%) and the pore size must be withina critical range (usually 70–200 µm). The porosity must be interconnected to al-low ingrowth of cells, vascularization and diffusion of nutrients. Recently, thefamily of poly(α-hydroxy acid)s such as polyglycolide (PGA), polylactide (PLA)and its copolymer poly(D,L-lactide-co-glycolide) (PLGA) are extensively used ortested for the scaffold materials as a bio-erodible material due to relatively goodbiocompatibility, controllable biodegradability and relatively good processability.Also, these polymers are easily formed into desired shapes with good mechani-cal strength [6–10]. However, most of polymeric scaffolds have hydrophobic sur-face which tend to unfavorably influence their cell-compatibility. So, surface hy-drophilicity of the scaffold is needed for optimal cell seeding, serum protein ab-sorption and cell attachment and in-growth [11, 12].

It is more desirable to make up for a fault for the PLA, PGA and PLGA scaffoldsfor the applications of tissue engineering. For example, hydrophobic surfacesof PLA, PGA and PLGA possess high interfacial free energy in aqueous solutions,which tend to unfavorably influence their cell-, tissue- and blood-compatibility ininitial stage of contact, so it might be more favorable to create a hydrophilic surface,resulting in uniform cell seeding and distribution [13–23]. Another example is thatthe bioactive material-impregnated scaffolds might be better for cell proliferation,differentiation and migration due to the stimulation of cell growing from thesustained release of cytokine molecules such as fibroblast growth factor (FGF),nerve growth factor (NGF) and vascular endothelial cell growth factor (VEGF) [24].

Small intestine submucosa (SIS), a natural tissue from porcine jejunum, is one ofthe significant natural bioactive materials. The SIS material consists of naturallyoccurring ECM that has been shown to be rich in components which supportangiogenesis such as fibronectin, glycosaminoglycans including heparin, severalcollagens including Types I, III, IV, V and VI, and angiogenic growth factorssuch as basic FGF (bFGF) and VEGF [25]. In addition, Badylak et al. describedsystematically that an acellular resorbable scaffold material derived from the SIShas been shown to be rapidly resorbed, support early and abundant new blood vesselgrowth and serve as a template for the constructive remodeling of several body tissueincluding musculoskeletal structures, skin, body wall, dura mater, urinary bladderand blood vessels [26–31].

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In this study, we developed the novel natural/synthetic hybrid scaffolds as the SISimpregnated PLGA (SIS/PLGA) so that polymeric scaffolds were given bioactivefunctionality of SIS material. Fabrication condition of the SIS/PLGA scaffoldswas optimized to produce the macroporous structure and high content of SIS inorder to maximum of bioactivity. All scaffolds were characterized by scanningelectron microscopy (SEM), mercury intrusion porosimeter. Degradation behaviorof the SIS/PLGA scaffolds was confirmed by measuring the weight loss in vitro.In addition, the effect of SIS from the SIS/PLGA scaffolds was observed by theimplantation subcutaneous onto the athymic nude mouse for ostoconductivity.

MATERIALS AND METHODS

Materials

Poly(D,L-lactide-co-glycolide) (PLGA; molecular weight 90 × 103 g/mol, 75 : 25by mol ratio of lactide to glycolide, Resomer® RG756) was purchased formBoehringer-Ingelheim (Ingelheim, Germany). Sections of porcine jejunum wereharvested from market weight pigs within 1 h of killing and prepared accordingto the method of Badylak et al. [32]. Briefly, the entire small intestine of pigswas removed and washed several times with saline and then it was cut into 10-cmstrips. Mesenteric tissue was removed and the intestine was everted. The mucosalepithelium and lamina propria were removed by gentle abrasion. The segmentwas then everted to normal orientation and the tunica serosa and tunica muscularisexterna were mechanically removed (Fig. 1A). Subsequently, SIS was sterilizedwith 0.1% peracetic acid and stored at −80◦C until use. The SIS dried to evaporatewater at 70◦C for 48 h using freeze dryer (Model FDU-540, Eyela, Japan). The driedSIS was pulverized using freezer mill (SPEX 6700, Mutchen, USA) at −198◦C to

(A) (B)

Figure 1. Schematic diagram of the preparation processing of SIS. (A) Section of porcine jejunumand SIS and (B) SEM microphotographs after freezer-milled SIS (original magnification ×100).

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get 10–20 µm size of SIS powder for the improvement of dispersivity into PLGAmatrix as shown in Fig. 1B.

Fabrication of the SIS/PLGA scaffolds

The SIS/PLGA scaffolds as natural/synthetic hybrid were fabricated by solvent cast-ing/salt leaching (SC/SL) method from mixtures composed of PLGA as biodegrad-able matrix, SIS powder and sodium chloride (NaCl; Junsei, Japan) as porogen.First, the NaCl particles sieved to a size range of 180–250 µm, 250–425 µm and425–600 µm, respectively, and then added to a solution of 20% (w/v) concentra-tion of PLGA in methylene chloride (MC, Junsei). Fabrication parameters of theSIS/PLGA scaffold, including PLGA, SIS powder and salt contents as shown in Ta-ble 1, were optimized to produce the desired foam microstructure and the maximumof SIS content. The SIS/PLGA scaffolds were air-dried for 48 h and subsequentlyvacuum-dried for 24 h to remove any residual solvent. The resulting SIS/PLGAscaffolds were immersed in deionized water for 48 h with changed every 6 h toleach out NaCl and then finally vacuum-dried. These totally dried scaffolds werestored in a desiccator under vacuum until use.

Characterizations of the SIS/PLGA scaffolds

Surface and cross-sectional morphologies of the SIS/PLGA scaffolds were observedby scanning electron microscopy (SEM, Model S-2250N, Hitachi, Japan) to inves-tigate the pore structure and pore size. Samples coated with gold for 1 min underargon atmosphere using plasma sputter (SC 500K, Emscope, UK). The SIS/PLGAscaffolds were analyzed by mercury intrusion porosimeter using an AutoPore II9220 (Micromeritics, USA) to determine pore size distributions, specific pore area,

Table 1.Processing variables, median pore diameter and porosity of the SIS/PLGA scaffolds

Code Mixture ratio Salt size Median pore PorositySIS/PLGA Salt/(PLGA+SIS) (µm) diameter (%)

(µm)

S1 0.4 8 250–425 69.23 90.70S2 0.8 8 250–425 97.64 94.06S3 1.0 8 250–425 71.98 92.79S4 1.2 8 250–425 90.46 93.39S5 1.6 8 250–425 77.91 89.14A1 1.6 6 250–425 70.02 72.99A2 1.6 8 250–425 82.20 91.29A3 1.6 10 250–425 88.69 95.21A4 1.6 12 250–425 90.46 98.39Z1 1.6 8 180–250 70.34 92.95Z2 1.6 8 250–425 90.46 94.39Z3 1.6 8 425–600 105.82 93.34

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median pore diameter and porosity. A solid penometer volume was ranged with6.7–7.3 ml and 0.1 g of sample was analyzed. Mercury was filled from a fillingpressure of 3.4 kPa and intruded to a maximum pressure of 414 MPa.

Wettability of the SIS/PLGA scaffolds

In order to compare the wettability of PLGA (control) and SIS/PLGA scaffolds,a 0.05% (w/v) red dye solution was dropped onto these scaffolds and then pho-tographs were taken at various times thereafter. In water absorption test, the scaf-folds were immersed in phosphate-buffered saline (PBS, Gibco-BRL, USA) atpH 7.4 for 30 s. Water absorption (WA, %) was calculated according to the fol-lowing equation:

WA = (Ws − Wo)/Wo × 100,

where Wo is the dry scaffold and Ws is the water absorbed scaffold. Water adsorptionin the short term was used to characterize the wettability of the scaffolds.

In vitro degradation

Each scaffold (Wo∼= 1000 mg) was suspended under aseptic conditions in PBS

(pH 7.4) containing 0.1% Tween 80 (Junsei) and 0.02% sodium azide (Sigma).They were incubated at 37◦C under a slow tangential agitation. Degradation wasfollowed for a period of 16 weeks. Samples were removed at regular time intervals,rinsed twice with distilled water, surface wiped. The samples were then freeze-dried overnight and weighed (Wr). Weight loss (%) of the scaffolds was calculatedaccording to the following equations:

Weight loss = (Wo − Wr)/Wo × 100.

The samples were weighed for each time point and weight loss was plotted againstthe incubation time.

BMSC attachment

To observe cell attachment in the SIS/PLGA scaffolds, human bone marrow stromalstem cells (BMSCs) were obtained from an adult volunteer (male, 24 years of age).Briefly, the 20 ml aspirate was diluted 1 : 1 Hanks’ balanced salt solution (HBSS,Gibco-BRL) and layered over 10 ml Percoll (Sigma). After centrifugation at 200×g

for 25 min, the mononuclear cell layer was recovered from the gradient interfaceand washed with HBSS. The cells were centrifuged at 50 × g for 10 min andresuspended in complete culture medium (Dulbecco’s modified Eagle’s medium;DMEM, Gibco-BRL). All the cells were plated in 15 ml medium in a 75-cm2 cultureflask (Falcon, USA) and incubated at 37◦C with 5% humidified CO2.

Scaffolds were pre-wet with 70% ethanol (Junsei) for 30 min and washed withPBS three times. Scaffolds were transferred to each well of a 24-well culture plate.

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At passage 3, the BMSCs were seeded at 5 × 106 cells/ml into the scaffolds. After1-day culture, mitochondrial dehydrogenase activity of BMSCs in scaffolds wasdetermined by using the water-soluble enzyme substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma), which was converted to bluewater-insoluble product formazan accumulated in the cytoplasm of viable cells.In brief, 200 µl of MTT solution (5 mg/ml) was freshly added to culture dishcontaining 2 ml of fresh medium and incubated at 37◦C and 5% CO2 for 4 h. Theintracellular formazan was solubilized using 2 ml of lysing buffer containing 45%dimethyl formamide and 10% sodium dodecyl sulfate. The absorbance of formazanproduced was measured at 590 nm with a Bio-Rad microplate reader (USA). Viablecell number was determined using a linear correlation between absorbance andBMSCs concentration.

Implantation of the SIS/PLGA scaffolds

To observe bone formation of the SIS/PLGA scaffolds, BMSCs were seeded at5 × 106 cells/ml into the scaffolds. After 1 day culture, scaffolds were implantedsubcutaneously under the dorsal skin of athymic nude mouse (Balb/C, CharlesRiver, Japan). The mice were killed after 4 and 8 weeks and then stained vonKossa staining for the confirming matrix mineralization. The samples fixed in10% buffered formalin (Sigma) and thin sections were cut from the paraffin-embedded tissue. Briefly, after we washed the samples multiple times withdeionized water, we added 5 ml 2% silver nitrate solution to the specimens. Treatedspecimens were placed in the dark for 15 min and then exposed to a bright light15 min. PLGA scaffold without implantation was also treated as negative control.Staining was evaluated as either positive or negative by two independent observers.Photomicrographs were taken using an Olympus light microscope.

Statistical analysis

The mean and standard deviations are reported for each experimental group. Sta-tistic analysis was performed by Student’s t-test (independent-difference). Resultswere considered significant at P < 0.05.

RESULTS

Fabrication and characterization of the SIS/PLGA scaffolds

In order to endow in new bioactive functionality from SIS as natural source to PLGAsynthetic biodegradable polymer, porous SIS/PLGA scaffolds as natural/synthetichybrid were fabricated by means of the SC/SL method. Scaffolds could befabricated into high porous, three-dimensionally interconnected structures. Theinfluence of SIS content, salt content and salt size on the scaffold characteristics iscompared. SEM images of the SIS/PLGA scaffolds with SIS/PLGA weight ratios

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Figure 2. SEM microphotographs of the SIS/PLGA scaffolds at different SIS contents ((A) S1,(B) S2, (C) S3, (D) S4 and (E) S5), different salt contents ((F) A1, (G), A2, (H) A3 and (I) A4)and different salt sizes ((J) Z1, (K), Z2 and (L) Z3) (original magnifications ×100).

of 0.4, 0.8, 1.0, 1.2 and 1.6 are shown in Fig. 2A–E. 1.6 g SIS and 1 g PLGA werefixed and then NaCl (250–425 µm of NaCl size) contents were varied with 15.6,20.8, 26.0 and 31.2 g, as shown in Fig. 2F–I. Figure 2J–L shows the effects ofthe different salt size (180–250, 250–425 and 425–600 µm of NaCl sizes) on thepore morphologies for the SIS/PLGA scaffolds. All of surface, cross-section andside of the SIS/PLGA scaffolds were highly porous with good interconnectionsbetween pores in which can support the surface of cell growth, proliferation and

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differentiation. Particularly, a uniform distribution of well-interconnected poresfrom the surface to core region due to the characteristics of the SL techniquewas observed. It can be observed that the pore size was almost the same,69.23–105.82 µm, as listed in Table 1.

Wettability of the SIS/PLGA scaffolds

Figure 3 shows the wettability of the SIS/PLGA scaffolds in comparison withcontrol PLGA scaffold. The drop of red dye solution was easily wetted in theSIS/PLGA scaffolds within 3 min (S1) and 30 s (S5) due to more hydrophillizedsurface by SIS. However, control PLGA scaffold did not wet for over 1 h. It couldbe observed that a completely wetted tissue-engineered scaffold was achieved withuniform cell seeding and in-growth into scaffolds.

Figure 4 compares the amount of water absorption by the scaffolds for 30 sin ambient atmosphere. Expectedly, the ability to absorb water increased withincreasing SIS content due to the hydrophilicity of SIS. The water absorption ofthe SIS/PLGA scaffolds showed significant differences (P < 0.05) in comparisonwith control PLGA scaffolds.

In vitro degradation

The weight loss degradation of PLGA scaffolds in PBS was measured up to 16weeks. As shown in Fig. 5, weight loss of all PLGA scaffolds slowly increased

Figure 4. Water absorption of the SIS/PLGA scaffolds with different SIS content (∗P < 0.05compared to control).

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Figure 5. Weight loss of the SIS/PLGA scaffolds along the incubation time; (2) PLGA (control),(1) S1, (Q) S2 and (P) S5.

until about 8 weeks of incubation time and then the weight rapidly decreased until16 weeks. After a 16-week period, the control PLGA scaffold lost over 90% ofits original weight. We observed that weight loss of PLGA scaffolds decreasedgradually with increasing SIS content. Degradation period of scaffolds is a veryimportant factor in tissue engineering when biodegradable scaffolds are used.

BMSC attachment

BMSCs are an important cell source for tissue-engineering applications. Also, itseems that surface properties of polymeric scaffolds are important for the initialstage of cell–polymer contact. As shown in Fig. 6, we observed that BMSCattachment increased gradually with increasing SIS content due to improvementof surface wettability of polymer scaffolds and good cell compatibility of SIS. Cellattachment of the SIS/PLGA scaffolds showed significant differences (P < 0.05)in comparison with control PLGA scaffolds.

Implantation of the SIS/PLGA scaffolds

BMSC-seeded scaffolds were utilized to transport and applied as a template guidingthe bone formation from the osteoinduction and ostetoconduction by SIS as natural

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Figure 6. BMSC attachment in the SIS/PLGA scaffolds with different SIS content (∗P < 0.05compared to control).

source. PLGA (control group) and SIS/PLGA (experimental groups S1 and S5)scaffolds were implanted on the subcutaneousness of athymic mice to investigatethe effect of SIS. The mice were killed after 4 and 8 weeks implantation. In PLGAscaffolds, there was no evidence of new bone formation at inner part of the PLGAscaffold (Fig. 7A, B). It might be suggested that the hydrophobicity of PLGA did notallow adhesion and migration of the BMSCs into the PLGA scaffold, resulting inthe bone formation of the vicinity of scaffolds. However, in the SIS/PLGA scaffold(Fig. 7C–F), we can observe the evidence of bone formation by the bioactivityof the SIS/PLGA scaffolds from the seeded BMSCs and undifferentiated stemcells in the subcutaneous sites and other soft connective tissue sites [33] havinga preponderance of stem cells compared with PLGA scaffold. Badylak and co-workers have successfully carried out the isolation and the identification of manykinds of secreted, circulation and extracellular matrix-bound growth factors fromSIS [34]. So, the explanation might be that these growth factors significantlyaffected the critical processes of tissue development and differentiation for boneformation. The SIS/PLGA scaffolds had significantly stronger positive expressionof the typical appearance of matrix mineralization than PLGA scaffold. Also,S5 (160% SIS impregnated) was stronger bone formation than S1 (40% SISimpregnated). This result could be caused by a variety of growth factors, cytokinesand ECM of SIS as natural source.

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Figure 7. Pictures of von Kossa staining of the (A, B) PLGA (control), (C, D) S1 and (E, F) S5 after(A, C, E) 4 and (B, D, F) 8 weeks implantation.

DISCUSSION

This paper reports novel natural/synthetic hybrid scaffolds to fabricate the macro-porous and three-dimensionally interconnected SIS/PLGA scaffolds for tissue-engineered bone. Fabrication parameters are SIS content, salt content and salt size.In addition, in order to maximize the effect of SIS material, we fabricated highcontent of SIS-impregnated PLGA scaffolds. However, over 160% of SIS weightover PLGA was not fabricated. It was observed that the size distribution of theSIS/PLGA scaffolds was constantly uniform, since the size distribution of a poro-

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gen as NaCl was the same, resulting in relatively large surface area per volume,higher porosity, almost the same interconnective structure between pore, more uni-form the pore size and pore size distribution, no change pore size and distributionvaried with SIS contents and less processing variables compared with other prepa-ration methods.

The surfaces of almost polymers are hydrophobic, which tend to unfavorablyinfluence their cell compatibility in the initial stage of contact. So, it is desirableto change their surface to hydrophilic. For instance, Mikos et al. used the ethanol-prewetting method [35] and Langer and co-workers investigated the effect ofNaOH treatment of PGA fibers on cell attachment [36] in order to overcomehydrophobicity. The wettability of the SIS/PLGA scaffolds was found to dependstrongly on the SIS content. Faster penetration of cell-culture medium, betteruniform cell seeding and distribution and better cell migration and growth mightbe expected.

Although many studies have been done on the degradation of PLGA scaffoldsin vitro and in vivo, the degradation period is difficult to predict because of thecomplication of the experimental conditions of the tissue-engineered scaffolds suchas scaffold characteristics (pore size, pore structure and porosity), and in vitro andin vivo environment. The physical and chemical requirements of ideal scaffoldsfor cell/tissue ingrowth are (i) biocompatibility, (ii) promotion of cell adhesion,(iii) enhancement of cell growth, (iv) retention of differentiated cell function,(v) large surface area per volume, (vi) highly porosity to provide adequate spacefor cell seeding, growth and ECM production and (vii) a uniformly distributes andinterconnected pore structure (this factor is very important so that cells are easilydistributed through the scaffolds and an organized network of tissue constituentscan be formed) [37].

We can observe the bone formation with the SIS/PLGA hybrid scaffolds comparedwith control PLGA scaffolds. Possible explanation is the exposure of SIS resultedin significant calcification as well as peri-implant fibrosis. It has been recognizedthat these peri-implant fibrosis can be altered the healing characteristics of SIS. Ac-cording to Badylak and co-workers, bone defect repair by SIS may be conductiverather than inductive [38]. The SIS/PLGA scaffolds are suitable for bone tissueengineering because these scaffolds have advantages of a natural (SIS) and a syn-thetic material (PLGA). It has been recognized that SIS contains many kinds ofnaturally-occurring secreted, circulating and ECM-bound growth factors, such asplatelet-derived growth factor (PDGF), epidermal growth factor (EGF), transform-ing growth factor (TGF), bFGF and VEGF, and ECM as fibronectin, glycosamino-glycans (GAG) including heparin, as well as several collagens including Types I, II,IV, V and VI. In addition, SIS has been widely used and tested for skin substitutes,a filling matrix for cartilage defects in clinic due to the improvement availabilitythrough the commercialization by DePuy and Cook companies.

These results lead to the conclusion that SIS plays an important role in boneformation. SIS has a variety of growth factors, cytokines and ECM that can promote

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bone formation, so this can also lead to bone in-growth within the SIS/PLGAscaffold. In addition, the SIS/PLGA scaffold as natural/synthetic hybrid possessesgood processibility and good wettability. Future works on the more detailedmechanism of osteoinduction and osteoconduction, such as calcium contents andalkaline phosphatase specific activity, labeling and detection of implanted cells, thethermal and mechanical properties of the SIS/PLGA scaffolds and its relative animalexperiment using BMSCs seeded the SIS/PLGA scaffolds, are in progress.

Acknowledgements

This work was supported by Korea Ministry of Commerce, Industry and Energy(N11-A08-1402-05-1-3). The authors gratefully acknowledge Mr. Ji Wook Jangand Ms. Phil Kyung Shin of the Pukyung National University.

REFERENCES

1. R. Langer and J. P. Vacanti, Science 260, 920 (1993).2. N. L’Heureux, L. Germain and F. A. Auger, Science 284, 1621 (1999).3. D. Ferber, Science 284, 422 (1999).4. S. J. Peter, M. J. Miller, A. W. Yasko, M. J. Yaszemski and A. G. Mikos, J. Biomed. Mater. Res.

43, 422 (1998).5. J. J. Yoo and I. W. Lee, Tissue Engineering: Concepts and Application. Korea Medical

Publications, Seoul (1998).6. D. J. Mooney, C. L. Mazzoni, C. Breuer, K. McNamara, D. Hern, J. P. Vacanti and R. Langer,

Biomaterials 17, 115 (1996).7. C. E. Holy, M. S. Shoichet and J. E. Davies, J. Biomed. Mater. Res. 51, 376 (2000).8. E. J. Frazza and E. E. Schmitt, J. Biomed. Mater. Res. Symp. 1, 43 (1971).9. X. S. Wu, in: Encyclopedic Handbook of Biomaterials and Bioiengineering, Vol. 1, p. 1015.

Marcel Dekker, New York, NY (1995).10. M. S. Rahman and T. Tsuchiya, Tissue Eng. 7, 781 (2001).11. R. P. Lanza, R. Langer and W. L. Chick, Principles of Tissue Engineering. Academic Press, San

Diego, CA (1997).12. E. Bell, Tissue Engineering: Current Perspectives. Birkhäuser, Boston, MA (1993).13. S. J. Lee, G. Khang, Y. M. Lee and H. B. Lee, J. Biomater. Sci. Polymer Edn 13, 197 (2002).14. G. Chen, T. Ushida and T. Tateishi, Mater. Sci. Eng. C 17, 63 (2001).15. S. J. Lee, G. Khang, J. H. Lee, Y. M. Lee and H. B. Lee, Polymer (Korea) 24, 877 (2000).16. G. Khang, H. B. Lee and J. B. Park, Biomed. Mater. Eng. 5, 245 (1995).17. J. S. Choi, S. J. Lee, G. Khang, I. Lee and H. B. Lee, Biomater. Res. 6, 17 (2002).18. G. Khang, B. J. Jeong, H. B. Lee and J. B. Park, Biomed. Mater. Eng. 5, 259 (1995).19. G. Khang, J. H. Jeon, J. W. Lee, S. C. Cho and H. B. Lee, Biomed. Mater. Eng. 7, 357 (1997).20. G. Khang, J. H. Jeon, J. C. Cho, J. M. Rhee and H. B. Lee, Polymer (Korea) 23, 861 (1999).21. G. Khang, S. J. Lee, J. H. Jeon, J. H. Lee, Y. M. Lee and H. B. Lee, Polymer (Korea) 24, 869

(2000).22. G. Khang, J. H. Lee, I. Lee, J. M. Rhee and H. B. Lee, Korea Polym. J. 8, 276 (2000).23. G. Khang, M. K. Choi, J. M. Rhee, S. J. Lee, H. B. Lee, Y. Iwasaki, N. Nakabayashi and

K. Ishihara, Korea Polymer J. 9, 107 (2001).24. G. Khang, J. M. Rhee, I. Lee and H. B. Lee, Polymer Sci. Tech. 12, 239 (2001).

Dow

nloa

ded

by [

Nor

th D

akot

a St

ate

Uni

vers

ity]

at 0

4:45

20

Aug

ust 2

013

Macroporous SIS/PLGA scaffolds 1017

25. S. L. Voytik-Harbin, A. O. Brightman, M. Kraine, B. Waisner and S. F. Badylak, J. Cell Biochem.67, 478 (1997).

26. M. C. Hiles, S. F. Badylak, G. C. Lantz, K. Kokini, L. A. Geddes and R. J. Morff, J. Biomed.Mater. Res. 29, 883 (1995).

27. S. F. Badylak, G. C. Lantz and A. C. Coffey, J. Surg. Res. 47, 74 (1989).28. M. Rosen, J. Ponsky, R. Petras, A. Fanning, F. Brody and F. Duperier, Surgery 132, 480 (2002).29. G. E. Sandusky, S. F. Badylak and R. J. Horff, Am. J. Pathol. 140, 317 (1992).30. R. O’Connor, J. N. Harding III and G. D. Steinberg, Urology 60, 906 (2002).31. T. G. Smith, III, M. Gettman, G. Lindberg, C. Napper, M. S. Pearle and J. A. Cadeddu, Urology

60, 931 (2002).32. S. F. Badylak, G. C. Lantz, A. Coffey and L. A. Geddes, J. Surg. Res. 47, 74 (1989).33. T. J. Owen, G. C. Lantz, M. C. Hiles, J. V. Vleet, B. R. Martin and L. A. Geddes, J. Surg. Res.

71, 179 (1997).34. B. P. Kropp, J. K. Ludlow, D. Spicer, M. K. Rippy, S. F. Badylak, M. C. Adams, M. A. Keating,

R. C. Rink, R. Birhle and K. B. Thor, Urology 52, 138 (1998).35. A. G. Mikos, M. D. Lyman, L. E. Freed and R. Langer, Biomaterials 15, 55 (1994).36. J. Gao, L. Niklason and R. Langer, J. Biomed. Mater. Res. 42, 417 (1998).37. G. Khang and H. B. Lee, in: Methods of Tissue Engineering, A. Atala and R. Lanza (Eds), p. 771.

Academic Press, New York, NY (2001).38. M. A. Suckow, S. L. Voytik-Harbin, L. A. Terril and S. F. Badylak, J. Invest. Surg. 12, 277

(1999).

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