decellularized placental matrices for adipose tissue engineering

Decellularized placental matrices for adipose tissueengineering

Lauren Flynn,1,2 John L. Semple,3,4 Kimberly A. Woodhouse1,2,4

1Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto,Ontario, Canada, M5S 3E52Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Toronto, Ontario,Canada, M5S 3G93Division of Plastic Surgery, Department of Surgery, University of Toronto, 100 College Street, Toronto, Ontario,Canada, M5G 1L54Sunnybrook and Women’s College Health Sciences Centre, 76 Grenville Street, Toronto, Ontario, Canada, M5S 1B2

Received 28 September 2005; revised 16 January 2006; accepted 9 February 2006Published online 1 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30762

Abstract: A tissue-engineered adipose substitute would beinvaluable to plastic surgeons for reconstructive, corrective,and cosmetic procedures. This work involves the design of ascaffold for soft tissue augmentation incorporating the de-cellularized extracellular matrix (ECM) of human placenta.We have developed a protocol to decellularize an intact,large segment (8 cm by 8 cm) of the human placenta. Tofacilitate the complete decellularization of the dense matrix,a system was designed to perfuse the required chemicalsinto the placenta via the existing vasculature. Followingprocessing, the original architecture of the placental ECMwas preserved, including an intact vascular network. Histo-logical, immunohistochemical, and scanning electron micro-

scopic analyses confirmed the removal of the cells and cel-lular debris and characterized the composition and structureof the matrix. In vitro cell culture experimentation showedthat the placental decellular matrix (PDM) could facilitatethe adhesion of primary human adipose precursor cells atearly time points. The PDM has great potential for use as ascaffold for adipose tissue engineering, as the placenta is arich source of human ECM components that can be readilyharvested without harm to the donor. © 2006 Wiley Period-icals, Inc. J Biomed Mater Res 79A: 359–369, 2006

Key words: adipose tissue engineering; extracellular matrix;decellularization; human adipose progenitors; stem cells

INTRODUCTION

Postoperative, congenital, and post-traumatic lossof the subcutaneous adipose tissue layer results in scartissue formation and deformity.1 Numerous recon-structive surgical procedures that require the use ofadipose tissue or fat substitutes rely on the transfer ofcomposite tissue flaps from the patient.2 The donorsite morbidity associated with such procedures is sig-nificant and can amount to millions of dollars per yearin hospitalization and days of lost work. There aremany limitations to the existing autogenous and allo-plastic reconstruction techniques used for the repair of

large volume tissue defects.3 Even in cases where onlysmaller volumes of adipose tissue have been lost, suchas following lumpectomy for breast cancer, �50% ofthese patients report tissue fibrosis, resulting in in-creased breast firmness and breast deformity.4 Theaim of tissue engineering strategies is to create func-tioning, healthy tissues that are fully integrated intothe host system. One general approach followed is todesign a supporting scaffold or artificial extracellularmatrix (ECM) with properties of the native tissues intowhich it will be incorporated.5 Ideally, the deviceshould include a porous, degradable scaffold, with alarge surface area to promote the adhesion of specificcells of interest while also facilitating the diffusiveexchange of nutrients and waste and the in-growth ofhost vasculature.6 Furthermore, this ECM substituteshould define the desired three-dimensional tissue ar-chitecture and have sufficient mechanical strength tosupport the development of new tissues.

The ECM is a dynamic, multifunctional structurethat is composed of a complex mixture of proteins,

Correspondence to: K.A. Woodhouse; e-mail: [email protected]

Contract grant sponsors: National Sciences and Engineer-ing Research Council (NSERC) of Canada, Province of On-tario through the Advanced Regenerative Tissue Engineer-ing Centre (ARTEC)

© 2006 Wiley Periodicals, Inc.

proteoglycans, and glycoproteins with diverse, tissue-specific composition and organization. In vivo func-tions of the ECM are wide-ranging and include themaintenance of cell structure and function, tissue andorgan morphogenesis, and wound healing.7 Decellu-larized ECM constructs have been shown to promotecellular infiltration and host integration, reduce scartissue formation, and depending on processing, trig-ger minimal immune response.8 ECM scaffolds candegrade within the body and promote matrix remod-eling, thereby augmenting the regeneration of dam-aged or missing tissues.

We are investigating the use of placental decellularmatrix (PDM) as an allogenic scaffold for soft tissueaugmentation and reconstruction. The primary func-tion of the placenta is to mediate the exchange ofnutrients and waste between the fetal and maternalcirculatory systems.9 The placenta has a maternalcomponent, the decidua basalis, and fetal components,the chorionic plate and villi. In the fetal components ofthe placenta, the large blood vessels of the chorionicplate, which originate from the umbilical cord, inter-connect with the fine capillary beds of the chorionicvilli.10 As three-dimensional matrix architecture hasbeen shown to stimulate angiogenesis in vitro, thepreservation of this vascular network during process-ing could be useful in the design of a vascularizedadipose tissue construct.11 The placenta is a richsource of ECM and basement membrane components.The matrices of human placenta and adipose tissuecontain similar types of collagen, including types I, III,IV, V, and VI.12,13

Previous work in our laboratory included a feasibil-ity study of the decellularization of human placenta.The investigation included the design of a preliminaryperfusion system to facilitate decellularization solu-tion delivery during the first phase of processing. Theperfusion system aids in overcoming the diffusionlimitations associated with decellularizing thick tis-sues in static systems by delivering the decellulariza-tion solutions throughout the placenta via the existingplacental vasculature. The aim of our current studywas to optimize the decellularization protocol to facil-itate the complete decellularization of an intact 8 cmby 8 cm portion of the placenta incorporating theentire thickness of the tissue, thereby creating a scaf-fold suitable for large volume reconstruction. The de-cellularization of such a sizable tissue block posed asignificant challenge and required the development ofa novel decellularization strategy including the mod-ification of the original detergent protocol to includelauroyl sarcosinate.

A secondary goal of our study was to conduct invitro cell culture experimentation to assess the suitabil-ity of the PDM as a scaffold for adipose tissue forma-tion. Adipocytes, with cytoplasmic lipid concentra-tions as high as 90%, are large, fragile, and highly

prone to ischemic cell death, precluding their use intissue-engineered constructs.14 However, the stromaof adipose tissue also contains fibroblast-like adiposeprecursor cells, including both stem cells and more-committed adipose progenitors, that do not containvisible cytoplasmic lipid and are far more resistant tomechanical damage and ischemia.15,16 These cells arehighly proliferative and, upon growth arrest, are ca-pable of differentiation into mature adipocytes both invitro and in vivo.17 For these initial trials, the PDMscaffolds were seeded with primary human adiposeprecursor cells isolated from subcutaneous abdominalfat. The scaffolds were visually assessed at early timepoints to determine if cellular adhesion to the pro-cessed matrix was supported.

MATERIALS AND METHODS

Materials

All decellularization chemicals were purchased fromSigma-Aldrich Canada (Oakville, Canada) and were used asreceived. Water was distilled and deionized using a Milli-pore Milli-RO 10 Plus filtration system at 18 M� resistance.

Placental procurement

Placentas were obtained, with informed consent, fromnormal-term, planned Caesarian-section deliveries at Sun-nybrook and Women’s College Health Sciences Centre;Women’s College Hospital site, Toronto, Canada. All pla-centas were inspected by the attending physician and re-leased to the study if pathological analysis of the tissues wasnot required. The placentas were transported to the labora-tory in Earle’s balanced salt solution supplemented witharginine (0.07 g/L), dextran (30 g/L; molecular weight35,000–50,000), heparin (2 USP U/mL), 1% phenylmethyl-sulfonyl fluoride (PMSF) protease inhibitor, and 1% antibi-otic/antimycotic (ABAM) solution within 30 min of harvest-ing. Research ethics board approval for this study wasobtained from Sunnybrook and Women’s College HealthSciences Centre (REB No. 9918).

Placental decellularization

Our redesigned perfusion system is shown in Figure 1.The perfusion system is based on the methodology previ-ously described in the literature for the investigation of drugtransport across the placenta during pregnancy.18,19 Twoperistaltic pumps were used to regulate the independentmaternal and fetal circulations. Each of the networks wasequipped with three separate reservoirs to aid in the sys-tematic perfusion of the multiple decellularization solutions.The solution flow rates were monitored by in-line flow

360 FLYNN, SEMPLE, AND WOODHOUSE

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

meters and the pressure within the fetal arterial line wasrecorded using a research grade blood pressure transducer(Harvard Apparatus). The entire system was housed withina water bath to maintain the temperature of the placenta andall perfusates at 37°C.

Immediately after transport to the laboratory, an intactand highly vascularized portion of the placenta wasclamped in position in a specialized holding apparatus that

isolated an 8 cm by 8 cm section of the organ. The excessplacental tissue outside of the device was excised. To facil-itate the perfusion of both the fetal and maternal compo-nents, two major fetal arteries within the chorionic platewere cannulated using 18-gauge angiocatheters and the de-cidua was perforated on opposite ends of the holder usingtwo blunt cannulae.

The holding apparatus was placed in the placental cham-ber, designed to immerse the clamped tissue in a bath of theappropriate decellularization solution (Fig. 2). This allowedfor the combined delivery of the decellularization agents viaperfusion and diffusion. The maternal portion of the pla-centa was placed downwards in the device to facilitate thepassive drainage of the perfusates and cellular debris. Thefetal and maternal cannulae were attached to the perfusionnetwork tubing at the commencement of the decellulariza-tion process. A pump, connected to an overflow valve on theplacental chamber, removed the waste solutions thatdrained from the placenta and accumulated during the per-fusion process. The solutions were not recirculated to pre-vent re-exposing the tissue to the cellular debris.

Each placenta was subjected to a multistep 18 day decel-lularization protocol. An outline of the protocol is detailed inTable I. During the first six days of processing, the perfusionsystem was used to deliver the appropriate decellularizationsolutions for 8 h during the day. At night and following thesixth day of processing, the placenta was removed from theperfusion chamber and was agitated in a sealed container ofthe appropriate solution and maintained in an incubator at37°C. For the perfusion, the fetal flow rate was 1.4 mL/min

Figure 1. The placental perfusion system. The apparatusallows for the systematic perfusion of the multiple decellu-larization solutions through independent fetal and maternalcirculations. The system is housed within a water bath tomaintain the temperature of the placenta and the perfusatesat 37°C.

Figure 2. a: A placenta clamped in the placental chamber during the perfusive phase of the decellularization process. b: Aclose-up of the fetal portion of the placenta, with angiocatheters positioned in two major vessels. c: A close-up of the maternalportion of the placenta, with inserted cannulae.

DECELLULARIZED PLACENTAL MATRICES FOR ADIPOSE TISSUE ENGINEERING 361


and the maternal flow rate was 4.3 mL/min to ensure suf-ficient flow to remove cellular debris while providing timefor the decellularization agents to be effective and avoidingexcessive waste. During the perfusion of the first decellular-ization solution (Solution A), the perfusates were equili-brated with gas blends to mimic the physiological gas bal-ance and prevent the contraction of the placental vasculatureduring the first stages of decellularization. The fetal perfus-ate was equilibrated with a 96% N2/4% CO2 gas blend andthe maternal perfusate was equilibrated with a 95% O2/5%CO2 gas blend.

The decellularization process involved the systematic de-livery of four different decellularization solutions, as well astwo stages of enzymatic digestion (Table I). Briefly, theplacenta was treated for the first 48 h in Solution A, ahypotonic tris buffer solution at pH 8.0. Next, the placentawas subjected to a 48-h extraction in Solution B, a 1% solu-tion of lauroyl sarcosinate with tris base and a high saltconcentration. Following this, the placenta was treated for 6days in Solution C, a 1% tris-buffered solution of lauroylsarcosinate. The placenta was then rinsed thoroughly andsubjected to enzymatic digestion with DNase (15,000 U TypeII from bovine pancreas) and RNase (12.5 mg Type III Afrom bovine pancreas) for 24 h. The placenta was thenextracted for 72 h in Solution D, a 1% solution of tris-buffered Triton-X100. A second enzymatic digestion wasperformed for the next 24 h and the protocol was completedwith a final extraction in Solution D for an additional 72 h.All decellularization solutions were supplemented with 1%

ABAM and Solution A and Solution B were also supple-mented with 1% PMSF.

Histological and immunohistochemicalcharacterization

Histological and immunohistochemical staining was con-ducted on representative pieces of the PDM that were fixedin 10% neutral buffered formalin for 24 h to confirm cellularextraction and elucidate the matrix composition. Hematox-ylin and eosin (H&E) staining was used to detect the pres-ence of residual nucleated cells or cell fragments. Methylgreen-pyronine (MGP) was used to assess the removal of allof the DNA and RNA. Masson’s trichrome characterized thecollagen structure of the PDM and confirmed the absence ofresidual cells or cell fragments. Movat’s pentachromestained for collagen and elastin within the matrix. Immuno-histochemistry using nova red as a substrate was used tolocalize human laminin and type IV collagen expressionwithin the PDM.

Scanning electron microscopy

Scanning electron microscopy (SEM) analysis was con-ducted on samples of the PDM at the end of processing toconfirm decellularization and to assess the structure of thematrix. Each sample was rinsed thoroughly in PBS and fixedin 2% glutaraldehyde for 24 h. After extensive rinsing, thesamples were frozen in liquid nitrogen and fractured on thecross section, prior to critical point drying. The dried matri-ces were mounted onto microscopy studs with carbon paintand sputter-coated in platinum. Micrographs were obtainedwith a working distance of 15 mm and an acceleratingvoltage of 10 kV on a Hitachi model S-570.

Adipose precursor cell culture

Fresh, sterile abdominal fat samples were obtained withinformed consent from patients at Sunnybrook and Wom-en’s College Health Sciences Centre; Women’s College Hos-pital site, Toronto, Canada, who were undergoing electivesurgery involving the abdomen. The samples were collectedin cation-free phosphate buffer solution (PBS) supplementedwith 20 mg/mL bovine serum albumin (BSA) and weredelivered to the laboratory on ice for processing within 2 hof harvest. A 1–5 g tissue sample was minced and washed inPBS to remove blood, serum, and oil from the fragments.The tissue was digested for 45 min under agitation at 37°C ina solution of 2 mg/mL collagenase type II in Kreb’s Ringerbicarbonate buffer (pH 7.4), supplemented with 3 mM glu-cose, 25 mM HEPES, and 20 mg/mL BSA. Following diges-tion, the sample was filtered through a 250 �m stainless steelfilter to remove any undigested tissue fragments. An equalvolume of Dulbecco’s Modified Eagle Medium and Ham’sF-12 nutrient mixture (DMEM:Ham’s F12), supplementedwith 10% fetal bovine serum, 100 U/mL penicillin, and 0.1

TABLE IThe Placental Decellularization Schedule

Day 1 – perfusiona Solution ADay 2 – perfusiona Solution ADay 3 – perfusiona Solution BDay 4 – perfusiona Solution BDay 5 – perfusiona Solution CDay 6 – perfusiona Solution CDay 7 – staticb Solution CDay 8 – staticb Solution CDay 9 – staticb Solution CDay 10 – staticb Solution CDay 11 Enzymatic digestion no. 1c

Day 12 – staticb Solution DDay 13 – staticb Solution DDay 14 – staticb Solution DDay 15 Enzymatic digestion no. 2c

Day 16 – staticb Solution DDay 17 – staticb Solution DDay 18 – staticb Solution D

Solution A (hypotonic Tris buffer solution, pH 8.0): 10 mMTris base, 5 mM EDTA, 1% PMSF, 1% ABAM; Solution B(detergent extraction no. 1, pH 8.0): 50 mM Tris base, 1.5 Mpotassium chloride, 5 mM EDTA, 1% lauroyl sarcosinate, 1%PMSF, 1% ABAM; Solution C (detergent extraction no. 2, pH8.0): 50 mM Tris base, 1% lauroyl sarcosinate, 1% ABAM.

aPerfusion for 8 h during the day and agitation overnightin 1 L of the solution.

bAgitation in 1 L of the solution, changed twice daily.cAgitation in 160 mL buffer (55 mM Na2HPO4, 17 mM

KH2PO4, 4.9 mM MgSO4 � 7H2O) supplemented with 15,000U DNase Type II, 12.5 mg RNase Type III A, and 1% ABAM.



mg/mL streptomycin, was added to the filtrate. The samplewas allowed to gravity sediment for 5 min and the buoyantmature adipocytes were removed by aspiration. The remain-ing fraction contained the stromal-vascular cell population,including the adipose precursor cell population. The sam-ples were centrifuged at 1200 � g for 5 min and resuspendedin erythrocyte lysing buffer (0.154 M NH4Cl, 10 mMKHCO3, and 0.1 mM EDTA in sterile deionized water) for 10min under agitation at room temperature. Following thisprocessing, the cells were pelleted, resuspended in completemedia, and filtered through a 100-�m nylon filter. The cellswere plated in tissue culture flasks at 30,000 cells/cm2 andincubated at 37°C with 5% CO2. After 24 h in culture, thecells were washed repeatedly with PBS to remove any non-adherent cells or cell fragments. The complete media waschanged every 2–3 days. To passage the cells, confluentcultures were trypsin-released (0.25% trypsin/0.1% EDTA),washed, counted, and replated in new flasks at 30,000 cells/cm2.

To differentiate the harvested cell population into matureadipocytes, the cells were cultured in media supplementedwith factors to induce adipogenic differentiation.20 Morespecifically, confluent cells (passages 2–5) were cultured for14 days in serum-free DMEM:Ham’s F12 media supple-mented with 15 mM NaHCO3, 15 mM HEPES, 33 �M biotin,17 �M pantothenate, 10 �g/mL transferrin, 100 nM cortisol,66 nM insulin, 1 nM triiodothyronine (T3), 100 U/mL pen-icillin, and 0.1 mg/mL streptomycin. For the first 3 days ofculture, 0.25 mM isobutylmethylxanthine and 1 �g/mL oftroglitazone were added to the media.

Cell seeding

Prior to seeding, the matrix was sectioned into samples bymass, with each scaffold consisting of a 300 mg portion ofprimarily the villous tree network. The scaffolds were de-contaminated by three 30 min rinses in ethanol, rehydratedwith three washes in sterile PBS, and incubated overnight inserum-free DMEM:Ham’s F12 media prior to cell seeding.Adipose precursor cells at passage 2 were trypsin-released

prior to confluence and stained with Cell Tracker™ Green.The cells were resuspended at a concentration of either 1.2 �106 or 1.2 � 107 cells/mL and 50 �L of cell suspension wasapplied to the top of the PDM scaffolds or collagen type Icontrol gels in 24-well plates. The first seeding density wasselected based on the number of cells for confluence intwo-dimensional culture and the second density was chosento investigate the effects of a higher cell population. Thesamples were incubated for 3 h prior to the addition ofgrowth media to facilitate cellular attachment. Confocal mi-croscopy (Zeiss LSM510, FLUAR 20x/0.75 NA objectivelens, excitation with an Argon laser at 488 nm) was used toinvestigate the cellular adhesion and organization in thesamples at 24 h, 72 h, and 7 days. All seeding experimentswere performed in triplicate.

RESULTS

Placental decellularization

A significant volume of blood was removed fromthe placental tissues during the perfusion processing.In this phase, the fetal and maternal regions that weresupplied by the largest of the fetal vessels began toturn yellow. However, the peripheral tissues re-mained brown in color because of the reduced numberand size of the blood vessels that supplied these areas.At the end of day 6, the placenta was removed fromthe holding apparatus and the passive decellulariza-tion phase was commenced. This phase was associ-ated with augmented decellularization of the tissueedges. Commencing on day 9, the decellularizationsolution became cloudy and the matrix became moreloose and began to turn white. By the final day ofprocessing, the solution was no longer cloudy and theentire matrix appeared loose and white (Fig. 3). Mac-roscopically it appears that only the fetal portions of

Figure 3. The (a) top and (b) side view of a decellularized 8 cm by 8 cm section of the placenta. The entire segment of thematrix appears loose and white. Fully hydrated, the PDM is �3 cm thick, depending on the size of the placenta.



the placenta, consisting of the chorionic plate andvillous tree, remain at the end of the processing.

Histological and immunohistochemical analysis

Representative histological images of the untreatedplacenta and of the PDM are shown in Figure 4. Onlythe pink staining characteristic of collagen was visiblein the H&E sections of the PDM. The effective removalof the cells and cellular debris from the matrix wasconfirmed using Masson’s trichrome and Movat’spentachrome staining. Masson’s trichrome stainingalso revealed that the collagen architecture of the pla-cental chorionic plate and villous tree extensions mac-roscopically appeared to be preserved during process-ing. No elastin was observed in the PDM with theMovat’s pentachrome staining. The MGP staining in-

dicated that RNA and DNA were not present in thematrix at the completion of processing.

The results of the immunohistochemical staining forlaminin and type IV collagen in the PDM are shown inFigure 5. Collagen IV is present in both the chorionicplate and the villous tree extensions of the PDM.Within the villous tree, collagen IV expression is lo-calized to the exterior surfaces of the villi and alongthe blood vessels that pass through the interior of thestructures. Interestingly, laminin is only presentwithin the chorionic plate of the PDM. The lamininmay have been more easily removed from the fragilevilli during processing.

SEM analysis

SEM analysis of the ultrastructure of the PDMshowed a multitude of large villi emerging from the

Figure 4. The histological analysis of the placenta prior to processing, of the PDM following the completion of decellular-ization, and of the placenta after treatment with SDS. The H&E, Masson’s trichrome, and Movat’s pentachrome stainingsindicate that there are no cells or cellular debris in the PDM following processing. The MGP staining shows that the PDM isfree of residual RNA and DNA. The sample treated with SDS shows macroscopic degradation of the matrix architecture, aswell as the presence of residual cellular debris. Original magnification �50.



chorionic plate and gradually branching into smallervilli [Figs. 6(a,b)]. No cells or cell fragments wereobserved on the external surfaces of these structures.The collagen in the chorionic plate and on the outsideof the villi appeared smooth. Cross-sectional images ofthe larger villi were obtained [Fig. 6(c)]. These villicontained numerous intact blood vessels. When exam-ined under higher magnification, successful removalof the endothelial cell layer was apparent [Fig. 6(d)].The collagen within the villi was woven to form adense supporting network. This matrix organizationmay impart both strength and flexibility to the PDM.The preservation of the vascular architecture may beof use in the creation of a vascularized adipose tissueconstruct.

Adipose precursor cell culture

The harvested adipose precursor cells in culture hada fibroblastic morphology [Fig. 7(a)] and proliferatedrapidly. When stimulated to differentiate through me-dia supplementation, a significant proportion of thecells accumulated intracellular lipid and assumed amultilocular phenotype [Fig. 7(b)] by day 7. The per-centage of the precursor cells that differentiated wasdependent on the sample source and declined withincreasing passage. Early-passage cells were used forthe cell seeding experimentation.

Cell seeding

Cell seeding density was shown to be a criticalfactor in the survival of the adipose precursor popu-

lation in both the PDM scaffolds and the collagencontrol gels. When the samples were seeded at thelower density of 60,000 cells per scaffold, the visual-ized cells had a rounded morphology at both the 72 htime point (Fig. 8) and the 7 day time point in all of thePDM and collagen samples. This rounded morphol-ogy correlated with dead cells in a live-dead assay(results not shown), indicating that the seeded cellswere dead by 72 h.

When the collagen gels were seeded at the higherdensity of 600,000 cells per scaffold, extended cellswith a fibroblastic morphology were visualized on thesurface at all time points. By 7 days, the entire surfaceof the collagen gel was covered with live adiposeprecursor cells. Moreover, the cells had begun to mi-grate into the first 100 �m of the control samples. Theseeded adipose precursor population strongly con-tracted the collagen gels, with the percent contractionincreasing with time. By the day 7 time point, thecontrol scaffolds had decreased in size by over 50%.

When the PDM scaffolds were seeded at 600,000cells per scaffold, live adipose precursor cells with afibroblastic morphology could be visualized in local-ized regions of the scaffold at both the 24 and 72 htime points (Fig. 9), indicating that the matrix couldsupport cellular adhesion. However, by day 7, all ofthe cells within the PDM samples had the roundedmorphology associated with dead cells. No contrac-tion of the PDM scaffolds was apparent at any of thetime points.

DISCUSSION

The development of a protocol to fully decellularizea large segment of the human placenta was extremelychallenging. Placentas are heterogeneous organs withvarying vascular architecture and density. Conse-quently, each organ responded to the treatment in aunique manner and decellularization occurred atvarying rates. During our previous investigation, theplacenta was only perfused for a few hours on the firstday of processing, following which it was sectionedinto 1 cm3 blocks for the remainder of the treatment.To develop a protocol to fully decellularize an intact 8cm by 8 cm tissue segment, we redesigned the perfu-sion system and extended the perfusive phase of thetreatment. Overall a successful and reproducible pro-tocol for the decellularization of the entire tissue sec-tion was developed.

While the original protocol for the decellularizationof small tissue blocks of the placenta relied on themild, nonionic detergent Triton-X100, the use of thisdetergent alone was not effective at fully decellulariz-ing the larger tissue samples, even over extended pe-

Figure 5. Immunostaining for human collagen IV andlaminin with nova red as a substrate. Collagen IV is ex-pressed both within the chorionic plate and the villous ar-chitectures of the PDM. In the villous portion, collagen IVexpression is localized to the external surfaces and along theblood vessels that pass through the interior. Laminin expres-sion is limited to the chorionic plate of the PDM. Originalmagnification �100.



riods of time. Hence, it was necessary to investigatethe use of alternative detergents.

In studies on arteries and bladders, decellulariza-tion with sodium dodecyl sulfate (SDS) was shown tobe complete and the matrix architecture was well con-served.21,22 However, SDS can interact strongly withcollagen, causing damage to more delicate tissues,23,24

and can be difficult to remove from the matrices fol-lowing processing. Residual SDS could limit the ap-plicability of the treated scaffolds for use both in vitroand in vivo, due to the cytotoxic nature of the deter-gent.25 When the placenta was treated with SDS (inplace of lauroyl sarcosinate in the described methods),the matrix swelled greatly, became gel-like and beganto visibly lose its original organization. Moreover, cel-

lular debris appeared to be trapped inside the matrix,with large pink and brown spots persisting in thestructure throughout the processing period. On thefinal day of treatment, following pH 9.0 rinses toremove the residual SDS, the placenta was sampledand sent for histological analysis. The results clearlyconfirmed that the decellularization was incomplete(Fig. 4). Furthermore, the damage to the collagen inthe villous trees was confirmed, with the completedestruction of the original architecture, including theblood vessel network. It was therefore concluded thatSDS was not a good choice of detergent for use in theplacental decellularization.

Lauroyl sarcosinate was selected as a detergent dueto the favorable results reported for its use in the

Figure 6. SEM characterization of the PDM. a: The ultrastructure of the PDM with the villi extending from the chorionicplate. b: The interconnected villous tree network of the PDM, with larger villi branching to form smaller villi. c: A cross sectionof the villi in the PDM, showing the intact matrix architecture and the preservation of the vascular network. d: A close-up ofa blood vessel within the villi showing that the endothelial cell lining has been removed, exposing the network-type collagen.



decellularization of various vascular structures.26 Thisanionic detergent was observed to augment decellu-larization of the placenta during the early phases ofprocessing. However, histological evaluation withMGP staining revealed that the detergent appeared tointerfere with the enzymatic digestion of the residualDNA and RNA in the matrix. Consequently, the final-ized protocol involved an initial extraction with thelauroyl sarcosinate to rapidly loosen the matrix andmaximize the initial cellular removal, followed bymultiple extractions with Triton-X100 to remove theresidual DNA, RNA, and cellular debris.

At the completion of the processing, the PDM wasreproducibly decellularized. Qualitatively the scaf-folds appeared to have good mechanical integrity andsurgical handlabililty. Once sterilized, the scaffoldscould be stored stably for extended periods of time in

sterile PBS. In solution, the matrices were observed tobe loose, white, and highly hydrated. The PDM rep-resents a rich source of ECM and basement membranecomponents for use in allogenic constructs. The har-vesting of placentas makes use of human tissues thatcan be harvested without harm to the donor and thatare commonly discarded.

The mesenchymal stem cell population present inthe stroma of adipose tissue represents an ideal cellsource for many tissue-engineering applications, in-cluding adipose tissue engineering.1 Liposuction tech-niques can be used to harvest autologous cells thatproliferate rapidly in culture and can be induced todifferentiate both in vitro and in vivo. For adiposetissue engineering, concerns associated with uncon-trolled differentiation in vivo would be limited as thecellular extraction and construct implantation sites

Figure 7. a: The adipose precursor cell population with the fibroblastic morphology characteristic of the cells prior todifferentiation. Original magnification �25. b: Adipose precursor cells differentiating into adipocytes with a multilocularmorphology stained with Oil Red O after 7 days in differentiation media. Original magnification �100. [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 8. The adipose precursor cells after 72 h in culture seeded at 60,000 cells on (a) collagen I gels or (b) 300 mg samplesof the PDM. The seeded cells have a rounded morphology that was associated with dead cells in a live-dead assay. Barsrepresent 50 �m.



could be matched within the subcutaneous tissuelayer. Experimental results indicate that during lipo-genesis, differentiating adipose progenitors activelysynthesize new ECM and are responsible for the reor-ganization of the matrix into the mature form found infat.27 Hence, it is possible that these cells could re-model the PDM into a structure more similar to that ofnative adipose tissue ECM. Additionally, adipose pre-cursor cells secrete bioactive factors when differenti-ating that stimulate endothelial cell growth and mo-tility in vitro and angiogenesis in vivo.28,29 Thesefactors could aid in the neovascularization of the en-gineered construct upon implantation. Promising invivo results have been obtained with preadipocytes inmice, where normal adipose tissue pads formed fol-lowing subcutaneous cellular injection.30

While the methods for the isolation and culture ofthe adipose precursor cell population have been de-scribed in the literature,31 further work needs to beconducted to optimize the growth and differentiationconditions for the cells in vitro. In our research, wehave observed that culture conditions, cell passage,and cell source significantly impact the ability of thecells to proliferate and differentiate. The best resultswere obtained when the cells were passaged prior toconfluence, were not frozen, and were used beforepassage 5. We investigated a number of different me-dia formulations for adipogenic differentiation. How-ever, the highest levels of differentiation were ob-tained with the adipogenic media described in theMaterials and Methods section. The inclusion of athiazolidinedione, such as troglitazone or rosiglita-zone, for the first three days of culture significantlyaugmented the number of precursor cells that differ-entiated into multilocular adipocytes.

In our initial investigation of the PDM as a scaffold

for adipose tissue formation, the scaffolds were shownto facilitate cellular adhesion at early time points (24and 72 h). However, by the 7 day time point, theseeded cells were nonviable. The inability of the PDMscaffold to support the long-term survival and prolif-eration of the seeded adipose precursor cells could berelated to the complex architecture of the PDM. PDMscaffolds have an extremely high surface area andtortuosity, and cell growth on these scaffolds could berestricted by the dispersion of the seeded cells. Cellsscattered throughout the matrix may have limited ex-posure to the contact-mediated cellular cues and para-crine factors that support cellular proliferation andsurvival. The investigation of higher seeding densitieswas contraindicated by the infeasibility of supplyingsufficient media within the 24-well plate molds tomeet the cellular demand.

We believe that the PDM has potential for use as ascaffold for adipose tissue formation. Future workwith the PDM scaffolds will focus on the in vitro andin vivo optimization and characterization of the seededadipose precursor cells in terms of cellular prolifera-tion, differentiation, gene expression, and protein ex-pression. One specific objective will be to investigatealternative culture conditions and seeding methods,including perfusive cell delivery via the placental vas-culature, to promote cellular attachment and viability.

CONCLUSION

The use of decellularized placenta as an allograftcould be a viable and economic alternative to existingtreatment strategies for soft tissue augmentation. Suf-ficient scaffolding material for large volume recon-

Figure 9. The adipose precursor cells seeded at 600,000 cells onto 300 mg samples of the PDM after (a) 24 h or (b) 72 h inculture. Bars represent 50 �m.



struction could be obtained from a single placenta,due to its size and rich matrix structure. Additionally,PDM can be easily sterilized, cut into a variety ofthree-dimensional shapes, and sutured in position. Aswell as replacing current repair methods, these con-structs could be used to promote regeneration in casesthat are typically left untreated, such as followinglumpectomy, where the creation of large voids resultsin scar tissue formation and subsequent loss of con-tour.

The authors acknowledge Mary Boyle RN and the deliv-ery suite staff at Sunnybrook and Women’s College HealthSciences Centre for their assistance with the placental pro-curement.

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decellularized placental matrices for adipose tissue engineering

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