Week 3 R Dilley Scaffolds - UWA€¦ · 12/08/15 7 5.’Combinaon’Scaffolds’! Utilisethebestqualitiesfromtwoor) moredevices)! Customtosuitpurpose! E.g.Combinationofhighstrengthof)

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Associate  Professor    Rod  Dilley  Dr  Rob  Marano  

Ear  Sciences  Centre  School  of  Surgery  

Harry  Perkins  Research  Building  4th  Floor  

Lecture  Outline  � History  � Purpose  � Functions  � Properties  � Approaches  to  bioscaffold  design    � Clinical  Example  � Video  

History  � Use  of  implants  dates  back  over  2000  years  � Modern  devices  began  in  late  1940’s  

�  British  Ophthalmologist  treating  fighter  pilots  �  Eye  injuries  containing  shards  of  canopy  plastic  healed  with  no  apparent  side  effects  or  reactions.  

�  Concluded  that  canopy  material  may  be  used  as  artificial  lens  (first  implanted  in  1949)  

�  Late  60’s  collaborations  between  engineers,  chemists,  biologists,  and  physicians,  led  to  formalizing  design  principles  and  synthesis  strategies  for  biomaterials  

Purpose?  � Used  in  tissue  engineering.  

� Overall  aim  of  developing  a  substitute  to  restore,  replace  or  regenerate  defective  tissues.  

� Bioscaffolds  +  cells  +  growth  stimulating  signals  (bioactive  molecules)  are  known  as  the  “Tissue  Engineering  Triad”.  

Func:on  of  Bioscaffolds  

� Mimic  the  form  and  function  of  the  extracellular  matrix  (ECM).  

Extracellular  Matrix  (ECM)  

� Almost  all  cells  are  anchorage  dependent  i.e  need  to  be  attached  to  something  …….ECM  

� ECM  has  multiple  components  and  is  tissue  specific.  

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ECM  � Five  broad  functions  

1.  Structural  support  and  physical  environment  for  cells  to  grow.  

2.  Provides  structural/mechanical  properties  3.  Provision  of  bioactive  cues  (cellular  

alignment).  4.  Acts  as  a  reservoir  of  growth  factors.  5.  Provides  a  changeable  environment  to  

allow  for  events  such  as  remodeling  and  neovascularisation  (wound  healing)  

ECM  

Han, D. & Gouma, P. I. Electrospun bioscaffolds that mimic the topology of extracellular matrix. Nanomedicine 2, 37-41 (2006).  

Scaffold  Proper:es  1.  Architecture  2.  Tissue  compatibility  3.  Bioactivity  4.  Mechanical  Properties    

Scaffold  Proper:es  1.  Architecture:    

� Void  volume  (vascularisation,  new  tissue  formation)  

� Porous  (metabolite  and  nutrient  transport)  

� Biodegradable  (degradation  rate  matching  that  of  neo-­‐tissue  formation).  

Scaffold  Proper:es  2.  Tissue  compatibility:  

� Cells  must  be  able  to  grow  upon  it  and  differentiate.  

� Scaffold  and  its  breakdown  products  must  be  non  toxic.  

Scaffold  Proper:es  3.  Bioactivity:  

� Able  to  interact  with  cells  to  regulate  activities.  �  Bioscaffold  may  include  certain  biological  cues  either  through  topography  or  through  the  presentation  of  bioactive  molecules.  

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Topography   Scaffold  Proper:es  4.  Mechanical  Properties:  

� Provide  shape  and  stability  to  tissue  defect.  

� Could  be  similar  to  that  of  the  host  tissue.  � Important  for  cell  differentiation.  

Approaches  to  Scaffold  Design  � Four  Main  Approaches  

1.  Pre-­‐made  porous  scaffolds  for  cell  seeding  

2.  Decellularised  extracellular  matrix  3.  Cell  sheets  with  secreted  ECM  4.  Cells  encapsulated  in  self  assembled  

hydrogel  

1.  Porous  Scaffolds  a.  Natural  

� Derived  from  biological  material  � Silk,  collagen,  alginates  etc  

b.  Synthetic  � Non-­‐biological  

� Glass,  ceramics  etc.  

a.  Natural  Scaffolds  � Autogenic/Autologous  

� Derived  from  the  patient  � Allogenic/Homogenic  

� Derived  from  different  individual  same  species  

� Xenogenic  � From  a  different  species  

a.  Natural  Scaffolds  � Advantages  

� Excellent  biocompatibility,  good  cell  attachment  etc.  

� Disadvantages  � Limited  physical  and  mechanical  stability  (not  suitable  for  load  bearing  situations).  

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b.  Synthe:c  � Greater  control  over  physical  and  mechanical  properties.  

� Inorganic  � Glass,  ceramics  (Hydroxyapatite)  

� Organic  � Polypropylene,  nylon,  teflon,  polystyrene,  polymethylmethacrylate  (plexiglas)  

b.  Synthe:c  � Inorganic        � Organic  

Glass   Hydroxyapatite  

http://www.engr.iupui.edu/~tgchu/myweb/photo.htm  

Teflon  arterial  stent  

Manufacture  � Process  of  manufacture  will  determine  the  final  properties  of  the  bioscaffold.  � Electrospinning    � Casting  � Nanoweaving  � 3D  printing  

Electrospinning  

http://www.ceramicnanofibers.com/technology.html  

http://coe.berkeley.edu/labnotes/0607/spinoff.html  

Kumbar, S. G., James, R., Nukavarapu, S. P. & Laurencin, C. T. Electrospun nanofiber scaffolds: engineering soft tissues. Biomed Mater 3, 034002 (2008).  

Wang, Y. et al. The synergistic effects of 3-D porous silk fibroin matrix scaffold properties and hydrodynamic environment in cartilage tissue regeneration. Biomaterials 31, 4672-4681 (2010).  

Cas:ng  

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Nanoweaving   3D  Prin:ng  

2.  Decellularised  ECM  � Generally  derived  from  an  allograft  or  xenograft.  

� All  cellular  components  are  removed.  � Left  with  the  ECM  � ECM  components  are  well  conserved  between  species.  

Decellularised  ECM  � Advantages:  

� Properties  are  perfect  for  homologous  functions  

� Also  useful  for  non-­‐homologous  functions  if    properties  are  similar.  

� Excellent  biocompatibility  � Disadvantages:  

� Poor  distribution  of  cells  when  seeding.  � Possible  immune  reactions  if  not  properly  decellularised.  

2.  Decellularised  ECM   3.  Cell  Sheets  � Cells  are  grown  on  a  specialised  surface  until  confluent.  

� Secrete  their  own  ECM.  � Cells  +  ECM  are  removed  from  the  culture  surface  as  a  single  sheet.  

� Can  be  stacked  into  multiple  layers.  

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3.  Cell  Sheets   3.  Cell  Sheets  � Advantages:  

� Secretes  own  ECM  � Rapid  neovascularisaton  � No  sutures  to  keep  in  place  

� Disadvantages:  � Limited  thickness  � Not  good  for  load  bearing  tissue  

3.  Cell  Sheets  

A. Lorenti, "Wound Healing: From Epidermis Culture to Tissue Engineering," CellBio, Vol. 1 No. 2, 2012, pp. 17-29.

4.  Cell  Encapsula:on  � Entrapment  of  living  cells  within  a  homogenous  solid  mass.  

� Most  recent  is  use  of  self  assembling  polymer  scaffold  from  liquid  monomers.  

� Can  suspend  cells  in  liquid  and  inject  into  defect.    

4.  Cell  Encapsula:on  � Advantages:  � Good  for  irregularly  shaped  defects  � Disadvantages:  � Not  good  for  load  bearing  tissues.  

4.  Cell  Encapsula:on  

Injectable hydrogel scaffold starts as a soluble liquid at room temperature, left, and forms a stable, nonshrinking gel, right, at body temperature after one minute. Tiffany N. Vo, Adam K. Ekenseair, Fred Kurtis Kasper, Antonios G. Mikos, Synthesis, Physicochemical Characterization, and Cytocompatibility of Bioresorbable, Dual-Gelling Injectable Hydrogels, Biomacromolecules, 2013,

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5.  Combina:on  Scaffolds  � Utilise  the  best  qualities  from  two  or  more  devices  

� Custom  to  suit  purpose  � E.g.  Combination  of  high  strength  of  synthetic  material  coupled  with  cell  growth  properties  of  natural  compounds  for  bone  regeneration.  

5.  Combina:on  Scaffolds  

SEM of the hybrid scaffold with composition of 50% bioactive glass and 50% PVA for comboscaffold. In  vitro  and  in  vivo  osteogenic  potential  of  bioactive  glass–PVA  hybrid  scaffolds  colonized  by  mesenchymal  stem  cells  Viviane S Gomide et al 2012 Biomed. Mater. 7 015004

Clinical  Example  � Tissue  Engineering  a  Tympanic  Membrane  

Cells   Scaffold  Bioactive    Molecules  

Silk  Fibroin  •  Fibroin  studied  with  biomedical  applications  – Biocompatibility  – Biodegradability  – Mechanical  properties    – Ability  to  form  diverse  morphologies  

Safety  and  compatibility  Growth  factors  Optimising  silk  designs   Silkworm

(Bombyx mori)

Silk  scaffolds   Overall  Goal  

Culture  of    TM  Cells  

Grow  on  bioscaffold  Repair  Hole  

Normal   Perforation  

Optimise  Scaffold-­‐Cell  Interaction  

Bioactive  Molecules  

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Silk  Fibroin  •  Silk  from  silkworms  or  spiders  

1.  Fibroin  (structural)  2.  Seracin  (glue-­‐like)  

•  Immunogenic  (e.g.  sutures)    

Silk from Bombyx mori cocoon

Degummed

Fibroin Remains

Solublised and Cast

Fibroin Membrane

Silk  Fibroin  •  hTM  cells  seeded  onto  SF  membranes    

 at  300  000  cells/well  

•  Cells  incubated  15  days  at  37oC    

Tissue  Engineering  A  TM  •  SEM  of  hTM  cells  on  SF  membrane  

Biocompa:bility/Degrada:on  

Middle  Ear  Cavity   Subcutaneous  

Effect  on  Perfora:on  Healing  

in the SFS and ACS groups appeared transparent andsmooth on day 28 (Fig. 2G, 2H). In contrast, control andpaper-treated TMs showed increased opacity, resemblingscar formation at the perforation site (Fig. 2E, 2F). Thesemi-quantitative otomicroscopic score in the SFS andACS groups were significantly higher than in the paperor control group (P < .05) at each of the time points, butwith no significant differences when compared to eachother (Table I). In terms of paper, although it showedhigher otomicroscopic scores compared to control groupat 7 days, there were no significant differences betweentwo groups at 14 and 28 days postoperatively.

Histological examination demonstrated obvious dif-ferences in the morphology of the neomembranes (Fig. 3).At 28 days, the TMs in the SFS and ACS groups showed awell-organized trilaminar membrane consisting of an

outer epidermal layer, a middle fibrous layer, and aninner mucosal layer, which was similar to the native TM.The TMs in both groups appeared normal with uniformthickness throughout (Fig. 3D, 3E). In contrast, the paperand control groups had thickened TMs at the perforationsite, mainly due to a disorganized fibrous layer (Fig. 3B,3C). It is noteworthy that part of the paper patch wasfound to incorporate into the regenerated TM in two ofthe animals at 28 days postoperatively. Numerous inflam-matory cells, predominantly lymphocytes, were observedaround the remaining graft, with scant giant cells, macro-phages, and eosinophils visible (Fig. 3F).

SFS and ACS Facilitated the Hearing Recoveryof Treated TM

ABR testing demonstrated that hearing recovery inthe SFS and ACS groups was significantly improvedcompared to the control group (Fig. 4). Auditory thresh-olds were similar in all guinea pigs measuredpreperforation (P > .05) and postperforation (P > .05).The average hearing threshold of all guinea pigs was18.2 6 0.3 dB; this significantly increased to 29.9 6 0.2dB after perforation, indicating that TM perforationcaused significant hearing loss (P < .001). Hearingthresholds in the SFS and ACS groups were not signifi-cantly different compared to the normal TM group fromthe earliest time point measured at day 7 (P > .05), sug-gesting that the animals underwent rapid hearingrecovery. By contrast, hearing thresholds in the controlgroup were still significantly worse than in normal TM,ACS, and SFS groups on day 28 (P < .05). In the papergroup, the hearing significantly improved when comparedto the control group on day 7 (P < .01). Although hearingthresholds were significantly worse when compared tothe other groups on day 14 (for normal and SFS, P < .05;for ACS, P < .01), the hearing thresholds in the papergroup were similar to the normal TM group on day 28.

TABLE I.Perforation Closure and Otomicroscopic Scores of Three Materials

at Different Time Points Following Grafting.

Group

PerforationClosure Otomicroscopic Score*

7days

14days

28days 7 days 14 days 28 days

Control0/6 3/6 6/6 0.33 6 0.21 2.83 6 0.48 5.33 6 0.21

Paper 4/6 6/6† 6/6 2.83 6 0.48§ 4.17 6 0.40 5.33 6 0.33

ACS 6/6‡ 6/6† 6/6 5.50 6 0.22§,††6.33 6 0.21§,††6.50 6 0.22§,††

SFS 6/6‡ 6/6† 6/6 5.50 6 0.22§,††6.17 6 0.17§,††6.83 6 0.17§,††

*Otomicroscopic scores (n 5 6) are presented as mean 6 standarderror of the mean (SEM).

†P < .05, control (spontaneous healing) vs. other groups (Fisher’sexact test).

‡P < .01, control vs. other groups (Fisher’s exact test).§P < .05, control vs. other groups (one-way ANOVA).††P < .05, paper versus ACS, or paper versus SFS (one-way

ANOVA).ACS 5 acellular collagen scaffold; SFS 5 silk fibroin scaffold.

Fig. 2. Otoscope images of guinea pig tympanic membrane (TM). Upper row shows (A) a TM perforation (asterisk) prior to grafting and fol-lowing repair with (B) paper, (C) acellular collagen scaffold (ACS), and (D) silk fibroin scaffold (SFS). The arrows in (B–D) indicate the graftsafter surgery. Lower row shows healed TMs 28 days postoperatively. The arrowheads indicate scar formation at the perforation site in (E)control and (F) paper groups. The healed TMs treated with (G) ACS and (H) SFS appeared transparent and smooth. [Color figure can beviewed in the online issue, which is available at wileyonlinelibrary.com.]

Laryngoscope 123: August 2013 Shen et al.: Tympanic Membrane Repair Using Silk and Collagen Scaffolds

1979

Reproduced  from  Shen, Y. et al. Laryngoscope (2013).  

Short  Video  � http://www.ted.com/talks/lang/eng/anthony_atala_growing_organs_engineering_tissue.html