A NOVEL HYALURONAN BIOMATERIAL (HYAFF®–11) AS SCAFFOLD FOR CHONDROCYTES AND BONE MARROW STROMAL CELLS

A NOVEL HYALURONAN BIOMATERIAL (HYAFF®-ll) AS SCAFFOLD FOR CHONDROCYTES AND BONE MARROW STROMAL CELLS ".I , M . F"IOrlm, ".I L • Scorzona, .I B . G" 1 G • L"" . .1 .., A . P'iacentmi rlgo I0, ISlgno1.1 I, A . F acchini 2 2 3 3 P. Gobbi , G. Mazzotti , M. Duca , A. Pavesio J

Laboratorio di Immunologia e Genetica, Istituto di Rieerea Codivilla Putti, Istituti Ortopedici Rizzoli, Via di Barbiano l/LO, 40136 Bologna,ltaly. 2 Unita

3

Complessa di Scienze Anatomiehe Umane e Fisiopatologia dell 'Apparato Locomotore, Via Irnerio 48. 40126 Bologna, Italy.

Fidia Advanced Biopolymers. Via Ponte della Fabbriea 3/11, 35031 Abano Terme (padova), Italy.

ABSTRACT Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a preformed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaffll> -II is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide succesful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaftl>-11: a) to maintain a chondrocyte phenotype when seeded with human chondrocytes; b) to induce osteoblast differentiation when seeded with rat bone marrow stromal cells (BMSC) in a mineralizing medium. Our data indicate that human chondrocytes seeded on Hyaftl>-11 express and produce collagen type 11 and downregulate the production of collagen type I. BMSC seeded on Hyaffll>-11 differentiate into cells positive for bone-specific markers. These results provide an in vitro demostration of therapeutic potential of Hyaffll>-II as a delivery vehicle in tissueengineered repair of articular cartilage and bone defects.

KEYWORDS Chondrocytes, bone engineering.

marrow stromal cells, hyaluronic acid derivative, tissue

INTRODUCTION Joint pain is a major cause of disability in middle-aged and older people. Pain usually results from degeneration of the joint's cartilage due to primary osteoarthritis or from trauma causing loss of cartilage'. Different techniques have been used to facilitate repair of articular cartilage such as debridemenr', drilling of subchondral bone'' and periosteal and perichondrial transplantation':'. On the other hand, bone injuries often require implantation of grafts, and although autogenous bone is the elective graft material, it is limited in supply and necessitates traumatic harvesting procedures". Allogenous bone is

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Application ofhyaluronan in tissue engineering

inherently limited by risks of rejection and/or disease transmission, whereas permanent synthetic grafts are limited by osteolysis and inflammatory reactions to wear debris/". Recent advances in biology and materials have pushed tissue engineering to the forefront of new treatments also for cartilage and bone repai?·IO. This technique combines isolated cells with scaffold/cell carriers in order to promote cartilage or bone formation and repair. A vast amount of research has been focused on the design of new scaffolds to improve the success of such therapeutical strategy. The ideal matrix should prevent cells from floating out ofthe defect, provide mechanical strength, make uniform celI spreading possible and stimulate in this case, the chondrogenic and osteogenic phenotypes ofthe transplanted celIs. Furthermore, it should be viscous enough to alIow three-dimensional trapping of celIs and adhesive enough to secure their fixation to the implantation site. Fibrin, polymers and polyglycolic and polylactic acids, alginate and collagen gels, are some examples of three-dimensional scaffolds used for cartilage repair ll - I S. On the other hand, poly-lactic acid, poly-glycolic acid, poly-propylene fumarate, and apatite are some examples of three-dimensional scaffolds which have been used for bone tissue engineering 16- 18 . Aim of the study was to evaluate in vitro the potential of Hyaff'll-ll, a recently developed hyaluronic acid-based biodegradable polymer, to maintain chondrocyte phenotype and facilitate mineralization of bone marrow stromal cells. MATERIALS & METHODS

Test material The scaffold used in this study was made of HyaffO-11 , a polymer derived from the total esterification of sodium hyaluronate (80-200 kDa) with the benzyl alcohol on the free carboxyl groups of glucoronic acid along the polymeric chain. The configuration used was a non-woven mesh that is a pad composed ofa random array of polymer fibers having a diameter of 40 urn and kindly provided by FAB. S.r.I. (FIDIA Advanced Biopolymers, Abano Terme, Italy).

Human chondrocytes isolation and seeding on the biomaterial Human articular cartilage specimens were obtained from the knees of3 patients aged 13, 18, 32 years undergoing joint replacement surgery. Fragments of the excised cartilaginous tissues were put in Dulbecco's Modified Eagle's Medium (DMEM) (GlBCO BRL, Grand Island, NY, USA) and chondrocytes were isolated by sequential enzymatic digestions: 30 min with 0.1 % hyaluronidase (Sigma, St. Louis, MO, USA», I hour with 0.5% pronase (Sigma) and 1 hour with 0.2% collagenase (Sigma) at 37°C in DMEM with 25 mM HEPES (Sigma), 100 units/ml penicillin (Biological Industries, Kibbutz, Israel), 100 ug/rnl streptomycin (Biological Industries), 50 ug/ml gentamicin (Flow Laboratoires, Biaggio, Switzerland), 2.5 ug/ml amphotericin B (Biological Industries). The isolated chondrocytes were filtered by 100 urn and 70 urn nylon meshes, washed, and centrifuged. The cell number and viability were assessed by the Tripan Blue dye exclusion method and the celIs were cultured under conventional monolayer culture conditions. Once sufficient celIs were available, usualIy after the 3'd th to 4 passage, chondrocytes were seeded onto 2x2 em HyaffB'-11 non-woven meshes at a density of Ixl0 6 celIs/cm 2 in 35 mm Petri dishes. Four different chondrocyte preparations were used and cultures were harvested at 1 h, 24 hrs, 7 days, and 14 days.

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For each experimental time point one sample was snap-frozen for immunohistochemical analysis and another one was processed for electron microscopy. Electron microscopy

Sterilized silicon wafer chips of 3 x 5 mm utilized as specimen holder were coiled up with a thin layer of sterile Hyaffll'-11 and on each device 2 x lOScells were deposed. The samples were then cultivated for 1 day in DMEM medium at 37° C and 5% C02. At the end of the growth period, the specimens were fixed with 1% glutaraldehyde in O. I M phosphate buffer pH 7.2 for 45 min., post-fixed in 1% osmium tetroxide in Veronal buffer for 30 min, dehydrated in an increasing ethanol series and critical point dried (Critical point dryer CPD 030, Bal-Tee AG, Lichtenstein). Before the electron microscopy analysis, all the samples were coated with a 1.5 nm thick Platinum - Carbon film (pt 80%; C 20%) by means of a multievaporation device Balzers MED 010 (BalTee). The observations were performed with a Field Emission In lens Scanning Electron Microscope (FEISEM) Jeol JSM 890 (Jeol LTD., Tokyo, Japan) at 7 kV accelerating voltage and 1 x 10-I 1 A probe current. Histochemistry and immunohistochemistry

Snap-frozen biomaterial scaffolds were sliced into 5 urn sections and stained with Aldan blue and Safranin-O. For immunohistochemistry, sections were predigested with O. I % of hyaluronidase (Sigma) and incubated with monoclonal antibody anti-collagen type II (Chemicon, Temecula, CA) for 1 hour at room temperature. Slides were then rinsed and incubated for 30 minutes at room temperature with secondary goat antimouse/rabbit antibody (Dako, Glostrup, Denmark), rinsed again and treated with newfucsin substrate detection Kit (Dako). Finally, samples were counterstained with hematoxylin. Negative control sections were obtained by omitting the primary antibody. Analysis ofmRNA expression by RT-PCR

Scaffold cultured cells were analyzed by semiquantitative RT-PCR in order to investigate temporal changes in collagen type I and II mRNA expression. mRNA was extracted by RNAzol B reagent (Biotecx Laboratories, Huston, TX) and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Perkin Elmer, Norwalk, CT) and oligo dT priming. A1iquots of cDNA were then amplified with collagen I, II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers in a Gene Amp PCR System 9600 thermocycler (Perkin Elmer) in conditions allowing a linear amplification range. Amplified products were electrophoresed on 2% agarose gel stained with ethidium bromide and compared with a 123 bp DNA ladder (Life Technologies Ltd, Paisley, UK) to confirm the predicted size. Relative levels of PCR products were quantified by densitometric analysis of gel photographs and normalized to the signal from the housekeeping gene GAPDH. Rat BMSC cultures and seeding on biomaterial

Bone marrow aspirates from inbred Fisher 344 rats femur (Charles River Laboratories, Wilmington, MA) were diluted and layered over Ficoll-Hypaque density gradient and centrifuged at 1300xg for 20 min. The nucleated cells were collected, washed twice and resuspended in a-J\1EM containing 15% heat inactivated FCS,

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freshly-prepared ascorbic acid (50/lg/ml) and antibiotics (lOO/lg/m1 penicillin G, 50/lg/ml gentamicin sulphate and O.3/lg/m1 Fungizone) (standard medium). They were then seeded on petri dishes at a concentration of 3xI04/cm2 . The media were changed twice a week and BMSC were allowed to grow until confluent. Cells were then trypsinized, tested for viability by eosin exclusion dye and finally seeded on non-woven Hyaftl' -11 mesh (2 x 5 x 2 mm ) at the density of 2x106 cells/em' in 200/l1 of standard medium supplemented with IOmM f3-GP (f3-glicerophosphate) and 1O·8M Dex (Dexametasone) (hereafter referred to as Dex + f3-GP medium) in 6-well petri dishes. After 5, 10, 20 and 40 days scaffold BMSC cultures and supernatants were collected and analysed for cell growth by MTT mitochondrial reduction test and differentiation by light and electron microscopy as above reported.

RESULTS & DISCUSSION Histochemistry and immunohistochemistry Chondrocytes grown on HyafFl9-ll appeared round in shape and most chondrocytes seem to adhere to the fibers as confirmed by FEISEM evaluation. A1cian blue and Safranin-O stain revealed the production of metachromatic extracellular matrix particularly at 14 days. Immunohistochemical analysis for collagen type II showed the re-expression of this molecule through the different incubation time evaluated, suggesting that chondrocytes grown on the biomaterial underwent to a progressive redifferentiation process.

Figure 1. Chondrocytes after the seeding (24 h) on Hyaff'-ll at FEISEM analysis. The cell adhesion on the bio-thread is evident. In correspondence to the nucleus, the cell shape is remarkably raised on the thread surface and the cell membrane is characterized by close and fine ondulopodia. On the contrary, other membrane areas are flattened on the thread and smooth. Not frequently, large ondulopodia are detectable at the border of the cell and the thread.

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mRNA expression of collagen type I and II RT-PCR was performed on scaffold cultured samples in order to follow up condrocyte re-differentiation at the mRNA level. Samples analyzed in the linear phase of amplification and visualized on agarose gel showed an evident signal modulation at the different time points. Collagen I mRNA expression decreased after 24 hours and was significantly down-regulated on days 7 and 14 (p
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Proliferation and differentiation of rat bone marrow stromal cells The MTT test, which was used to evaluate cell growth, indicates that cell proliferation peaked as early as day 10 (Fig.4). On day 40, mineralised areas of Dex + f3-GP medium cell cultures were strongly positive to von Kassa staining. Alkaline phosphatase showed a stronger staining only on the outer layer of the cells. Electron microscopy analysis showed that starting from day 20 the extracellularmatrix presented mineralised areas localised around the cells and also between the cells and the Hyatf'-ll fibres. These cells showed -osteoblastic features: a large ovoid nucleus and extensive granular endoplasmic reticulum. Large mineralised areas were observable on day 40 between osteoblasts and Hyatf'-II fibres (Fig. 5). Lacunae containing cells with typical osteocytic morphology could also be seen. Cell proliferation (MTT test)

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Figure 5. Electron microscopy of rat BMSC cultured on Hyatf'-11 for 40 days. H = Hyatf'-11 fiber, OB = osteoblast, OC = osteoclast. Magnification x6000.

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CONCLUSIONS In this study we showed that Hya~-11 scaffolds are suitable delivery vehicles for chondrocytes and bone marrow stromal cells. These cells are able to re-express their differentiated phenotype as in the case of chondrocytes or to differentiate in osteoblasts expressing osteogenic markers once they are grown in this three dimensional configuration. In fact, phenotypic instability of chondrocytes when they are removed from their cartilage matrix has been consistently observed in different animal species I9 ' 21. It has been shown that when chondrocytes are grown in monolayer cultures, they proliferate but they cease to express the specialised ~roteins of cartilage, like collagen type II, and become fibroblastic in appearancev" 3. This situation can be reversed using suspension cultures which promote the re-expression of cartilage phenotype24,2s . When rat BMST were cultured on Hya~-11 light and electron microscopy clearly demonstrated that after at least 20 days all the stages characteristic of the mineralization processes occurred. These in vitro data demonstrate that the association of osteoprogenitor cells with Hya~-11 can provide an appropriate carrier vehicle for repairing small bone losses such as nonunions and cavitational defects that can readily be filled.

ACKNOWLEDGEMENTS This work was supported by grants from IRCCS "Istituti Ortopedici Rizzoli", Bologna, C.N.R. Progetto Finalizzato "Materiali speciali per tecnlogie avanzate II" and Progetto Finalizzato Ministero della Sanita.

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