Week 3 R Dilley Scaffolds - UWA€¦ · 12/08/15 7 5.’Combinaon’Scaffolds’!...
Transcript of Week 3 R Dilley Scaffolds - UWA€¦ · 12/08/15 7 5.’Combinaon’Scaffolds’!...
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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