Designing natural bases scaffolds for cartilage tissue engineering applications

Transcription

1 Designing natural bases scaffolds for cartilage tissue engineering applications Vitor M. Correlo 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Taipas, Guimarães, Portugal ICVS/3B s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

2 U. Minho, Portugal - Braga & Guimarães BRAGA TAIPAS GUIMARÃES U. MINHO

3 What are we doing? Bone Cartilage Skin IVD Tendons Meniscus CNS Tissue engineering & Regenerative medicine Biomaterials Natural polysaccharides: Starch; Chitosan; Carragenan; Alginates; Ulvan; Hyaluronic acid; Gellan gum; Chondroitin sulfate; Natural, protein based: Silk fibroin; Marine collagen; Soy bean; Natural mimics: Synthetic peptides Cells Cell lines: SaOs-2, L292; etc... Stem/progenitor cells: Bone Marrow; Adipose Derived; Amniotic Fluid/membrane; Umbilical Cord; Blood and derivatives; hesc Primary cells: Fibroblasts, Chondrocytes, Keratinocytes, Endothelial cells

4 A TRULLY MULTIDISCIPLINARY GROUP Chemistry Medicine Biotechnology Materials Science 3B s Biology Materials Engineering NATURE Life Sciences

5 European Institute of Excellence on Tissue Engineering and Regenerative Medicine - Headquarters Headquarters and the Home for 3B s Research Opened in July 2008, AvePark, Taipas - Guimarães, Minho, Portugal * CEO Rui L. Reis 3600 m 2 new state of the art full equipped facility specially designed for TERM research

6 Our facilities The Institute's laboratory facilities occupy 3600 m 2 including: Chemistry labs Processing labs Characterisation labs Cell culture labs Microscopy labs Histology labs Bioreactor labs Animal testing facilities

7 Tissue Engineering (simplistic scheme) Cells Medium Scaffolds Growth Factors

8 polyanionic neutral Natural based polymers at 3B s polysaccharides chitin polycationic starch dextran chitosan hyaluronic acid carragenan (k, i, l) alginate carboxylated sulfated chondroitin sulfate ulvan proteins silk fibroin gellan gum polyesters collagen fibronectin poly(hydroxybutyrate) J.F. Mano +, J.R.Soc.Interface 07

9 STEM/PROGENITOR CELLS SOURCES Formal protocols with Hospitals and other Health Care Institutions: Centro Hospitalar do Alto Ave Stem/progenitor cells sources: Collaboration with other Universities (Medical and Veterinary Sciences): Bone Marrow (Rat, Rabbit, Goat, Human; mouse) Adipose Tissue (Rat, Rabbit, Goat, Human) Amniotic Fluid / Membrane ( Human) Umbilical Cord (Human) Blood (and derivatives)( Human)

10 What does distinguish us? In house facilities for FULL biomaterial development Material extraction & purification Material characterisation Material processing Cell characterisation Cell expansion Cell seeding In vitro testing In vivo testing Cell isolation

11 THE MAIN TISSUES WE TRY TO ENGINEER The 3B s Research Group is developing innovative strategies aiming at regenerating: Bone Cartilage Osteochondral defects Skin Intravertebral disc (IVD) Neurological tissues (with ICVS UM) Tendons Meniscus

12 TYPES OF CARTILAGE 2.ny.us/webpages/lmiller /photos/636532/cartilag e%20types.bmp

13 ARTICULAR CARTILAGE Avascular tissue that covers the joint Provides a low friction gliding surface Acts as a load-bearing and wear control structure Composed by chondrocytes and a dense extracellular matrix (ECM)

14 ARTICULAR CARTILAGE STRUCTURE Matrix component tightly organized Adapted from van Blitterswijk, Tissue Engineering, 2008 Structure dependent on the collagen fibers alignment

15 ARTICULAR CARTILAGE CHARACTERISTICS Avascular: lack of nutrient supply No speciallized cells in cartilage remodeling Low metabolic activity Inability of bone marrow MSCs or resident chondroprogenitor cells to generate hyaline cartilage Adapted from Huey et al., Science, 2012 Lacks ihnate abilities to mount a sufficient healing response

16 ARTICULAR CARTILAGE DAMAGE AND DISEASES Rheumatoid arthritis Osteoarthritis Traumatic accident or injury Wear and tear over time Treatments include medication and/ or surgery: Microfracture Autologous Chondrocyte Implantation (ACI) Matrix-Induced Chondrocyte Implantation (MACI) ng-outdoors/5-ways-to-avoid-kneeinjuries-while-running/

17 CARTILAGE TISSUE ENGINEERING CELLS SCAFFOLDS CULTURE CONDITIONS Chondrocytes Natural or synthetic materials Dynamic Stem cells from different sources Different processing techniques Hypoxia; Co-cultures. BIOACTIVE AGENTS

18 SCAFFOLD MATERIALS FOR CARTILAGE TE NATURAL SYNTHETIC Starch Polycaprolactone (PCL) Chitosan Poly lactic acid (PLA) Silk Poly(butylene) succinate (PBS)

19 SCAFFOLDS PROCESSING

20 Collagen type II Alcian Blue Toluidine Blue CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS CPBS bovine articular chondrocytes histological analysis after 28 days Alves da Silva et al., Acta Biomaterialia, 2009

21 Collagen type II Alcian Blue Toluidine Blue CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS CPBS 20 x bovine articular chondrocytes histological analysis after 28 days Alves da Silva et al., Acta Biomaterialia, 2009

22 Collagen type II Alcian Blue Toluidine Blue CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS CPBS 20 x bovine articular chondrocytes histological analysis after 28 days 20 x Alves da Silva et al., Acta Biomaterialia, 2009

23 Collagen type II Alcian Blue Toluidine Blue CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS CPBS 20 x bovine articular chondrocytes histological analysis after 28 days 20 x 20 x Alves da Silva et al., Acta Biomaterialia, 2009

24 Safranin O Toluidine Blue CARTILAGE TE USING CHITOSAN-BASED SCAFFOLDS CPBTA human mesenchymal stem cells isolated from bone marrow aspirates 28 days 20 µm 200 µm AGGRECAN COLLAGEN II COLLAGEN I SOX9 COLLAGEN X RUNX2 20 µm 200 µm Alves da Silva et al., JTERM, 2011

25 CARTILAGE TE USING NANOFIBER MESHES Collagen type II PCL using bovine articular chondrocytes Toluidine Blue Alcian Blue 28 days 50 µm 50 µm 100 µm 100 µm Alves da Silva et al., Tissue Engineering, 2009

26 CARTILAGE TE USING NANOFIBER MESHES Collagen type II SPCL using bovine articular chondrocytes 28 days 50 µm 100 µm Toluidine Blue Alcian Blue 100 µm 100 µm Alves da Silva et al., Tissue Engineering, 2009

27 BIOREACTOR STATIC CARTILAGE TE USING PCL NANOFIBER MESHES CULTURED ON A FLOW PERFUSION BIOREACTOR TOLUIDINE BLUE SAFRANIN O 100 µm 100 µm 50 µm 50 µm AGGRECAN COLLAGEN I COLLAGEN II 100 µm 100 µm 50 µm 50 µm hbmscs SOX9 COLLAGEN X RUNX2 28 Days Alves da Silva et al., Biomacromolecules, 2010

28 PCL NFM MODIFICATION WITH CHONDROITIN SULFATE 20 µm CARTILAGE-RELATED GENES ECM PRODUCTION Piai J, Alves da Silva et al., Submitted, 2014

29 CS-Immobilized PCL NFMs PCL NFMs MORPHOLOGICAL ANALYSIS 14 Days 21 Days 28 Days

30 CARTILAGE ENGINEERING Adapted from Kang et al., Biomaterials, 2012

31 CARTILAGE CO-CULTURES CONCEPT Paracrine signalling of chondrocytes will influence the stem cells chondrogenic differentiation The secreted molecules/ proteins provide a rich culture medium able to influence chondrogenesis without the addition of growth factors CONDITIONED MEDIUM

32 STEM CELLS CULTURES Cell source Articular cartilage Indirect co-culture using conditioned medium from human articular chondrocytes (hacs), without growth factor supplementation Dynamic cell seeding in a rotator during 24 hours Bone marrow hbmscs hwjscs CPBS fiber meshes Static culture during 4 weeks Umbilical cord Wharton s Jelly Alves da Silva et al., JTERM, 2013

33 GLYCOSAMINOGLYCAN PRODUCTION AND CARTILAGE-RELATED GENES EXPRESSION hwjscs produced significantly higher amounts of GAGs when compared to hbmscs. Cultures using hwjscs expressed higher levels of cartilage related genes compared to hbmscs. Alves da Silva et al., JTERM, 2013

34 CONCLUSIONS CS-immobilized PCL NFMs represents a better substrate for the maintenance of hacs phenotype, namely its typical round shape and cellular agglomeration/ clustering, without affecting cells viability, proliferation and ECM production. Therefore, CS-functionalized electrospun nanofibers represent a valuable substrate for culturing human articular condrocytes, envisioning a cartilage tissue engineering application.

35 Silk Fibroin-Based Scaffolds, Hydrogels and Calcium-Phosphate Filled Materials Aimed for Regenerative Medicine Applications Le-Ping Yan Doctoral Program in Tissue Engineering, Regenerative Medicine and Stem Cells Supervisors: Prof. Rui L. Reis, Dr. J. Miguel Oliveira and Dr. Ana L. Oliveira 1 3B's Research Group Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, S. Cláudio de Barco, Taipas, Guimarães, Portugal. 2 ICVS/3B s, PT Government Associated Laboratory, Braga/Guimarães, Portugal. 2014

36 Background: Defects in skeletal tissues Large defects in skeletal tissues are common problems in clinics Bone fracture Cartilage lesion Osteochondral defect (OCD) es/rss/ _s ar-cartilage-problems-knee 4_1.htm Current clinical strategies Autograft Allograft ADVANTAGES Desirable clinical outcome DISADVANTAGES Autograft: limited supplies; donor site morbidity; Allograft: risk of disease transmission.

37 Silk fibroin Spider silk Bombyx mori cocoon Main sequence of silk fibroin Main amino acid in silk fibroin Antheraea cocoon Advantages Easy access Biocompatible Biodegradable Tunable properties Non-woven net Porous scaffold 500 µm Biomaterials 2005, 26: Silk fibroin Hydrogel Film 1 cm µm Biomaterials 2004, 25: Microspheres Advanced Functional Materials 2005, 8: Biomaterials 2010, 31:

38 Development of robust silk fibroin scaffolds and used them as a platform for the following studies Aqueous derived salt-leaching approach Kim UJ... Kaplan DL. Biomaterials, 2005, 26, Advantages: Interconnected porous structure; All aqueous procedures; Environmentally friendly Disadvantage: Silk scaffolds cannot be prepared with more than 10% silk solution

39 Silk scaffold: Preparation procedure Cocoon Silk fibroin Concentrated silk solution Degumming Dissolution and dialysis Concentration by PEG solution Scaffolds prepared with 8, 10, 12 and 16% solutions were named as silk-8, silk-10, silk-12 and silk-16, respectively. Addition of NaCl particles Extraction in water Silk-8 Silk-10 Freeze-drying Silk-12 Silk-16 3 mm Yan LP Reis RL. Acta Biomater. 2012, 8(1): Scaffolds in wet status

40 Silk-NanoCaP scaffold: Preparation procedure 16% silk solution Silk-NanoCaP suspension Addition of CaCl 2 and (NH 4 ) 2 HPO 4 solutions ph 8.5, aging for 24 hours Addition of NaCl particles Salt-leaching/freeze-drying Groups Theoretical CaP content (%) Silk/CaP-4 4 Silk/CaP-8 8 Silk/CaP Silk/CaP Salt leached silk/cap scaffolds Silk/CaP-4 Silk/CaP-8 3 mm 3 mm Silk/CaP-16 Silk/CaP-25 3 mm 3 mm Yan LP Reis RL. Nanomedicine 2013, 8(3):

41 Silk-NanoCaP scaffold: 3D reconstruction (Micro-CT) Homogeneous distribution of CaP at macroscopic level Micro-CT image of pure CaP distribution Micro-CT image of CaP distribution in silk Silk/CaP-4 Silk/CaP-8 Silk/CaP-4 Silk/CaP-8 Silk/CaP-16 Silk/CaP-25 Silk/CaP-16 Silk/CaP-25 3 mm 3 mm Grey: CaP phase White: CaP phase; Grey: Silk matrix Yan LP Reis RL. Nanomedicine 2013, 8(3):

42 Development of robust and biomimetic silk based scaffolds for OCD regeneration Chondral layer: collagen, glycosaminoglycan Subchondral layer: spongy bone Osteochondral tissue le.aspx?id=14#.u3seopk7uso

43 Bilayered scaffold: Scaffolds preparation and study design Silk-NanoCaP suspension NaCl particles Silk solution NaCl particles Dry for 2 days Salt-leaching overnight Bilayered Silk/Silk-NanoCaP scaffold Pores inside the scaffolds Physicochemical characterization RBMSCs rabbilt bone marrow mesenchymal stromal cells Culture with RBMSCs Subcutaneous implantation Implantation in knee OCD Yan LP Reis RL. Submitted (2), 2014.

44 Bilayered scaffold: 3D reconstruction (Micro-CT) Integrated structure with distinct phase distribution Micro-CT image of the scaffold Micro-CT image of the pure CaP Brown: Silk matrix Blue: CaP phase 4 mm 4 mm Porosity distribution (%) Porosity distribution Length (mm) CaP distribution (area %) CaP distribution Length (mm) Yan LP Reis RL. Submitted (2), 2014.

45 Bilayered scaffold: Mechanical evaluation Superior mechanical properties Compressive modulus (MPa) Compressive modulus (MPa) 20 Dry status S16 SC16 Bilayered 0.5 Wet status n=6 n= S16 SC16 Bilayered E' (MPa) Storage modulus (DMA) S16 SC16 Bilayered Frequency (Hz) S16: Silk-16 SC16: Silk/CaP-16 Yan LP Reis RL. Submitted (2), n=5

46 Bilayered scaffold: RBMSCs viability and differentiation Silk-NanoCaP layer induced higher ALP activity of RBMSCs * p<0.05 Tukey s test ALP activity (µmol/hour/µg DNA) S16.Basal ALP: alkaline phosphatase 1 Week 2 Week S16.Osteo Normalized ALP activity * SC16.Basal * SC16.Osteo Cart.Basal Bone.Basal Cart.Osteo Bone.Osteo Bilayered.Basal RBMSCs were seeded onto the scaffolds and underwent osteogenic differentiation for 1 and 2 weeks * * RBMSCs: rabbilt bone marrow stromal cells * Bilayered.Osteo n 9 Absorbance (490 nm) * p<0.05 Yan LP Reis RL. Submitted (2), MTS assay * * Time (day) Tukey s test n 9

47 Bilayered scaffold: OCD regeneration in rabbit knee model Regeneration of osteochondral defect in rabbit knee Masson s trichrome staining Defect with scaffold S Scaffolds were implanted into rabbit critical size OCD (Ø 5 mm) for 4 weeks Cross-section of Silk/Nano-CaP layer S S 200 µm NB: New bone S: Scaffold Defect control S NB 200 µm 1 mm Yan LP Reis RL. Submitted (2), 2014.

48 Bilayered scaffold: OCD regeneration in rabbit model Regeneration of cartilage in osteochondral defect Safranin O staining S S S: Scaffold 200 µm Collagen II immunohistochemistry staining Control 200 µm Yan LP Reis RL. Submitted (2), 2014.

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