This document summarizes research on hydrogels and their applications in tissue engineering, regenerative medicine, and wound healing. It describes several key findings:
1) Hydrogels from konjac glucomannan were shown to stimulate proliferation of fibroblasts and keratinocytes in a concentration-dependent manner and support their metabolic activity.
2) Crosslinking konjac glucomannan formed hydrogels that maintained viability of fibroblasts and keratinocytes. The hydrogels inhibited contraction and promoted re-epithelialization in a human tissue engineered skin model.
3) Analysis found the hydrogels modulated water content and interactions to influence cell-matrix interactions important for wound healing and tissue regeneration applications.
Study on Air-Water & Water-Water Heat Exchange in a Finned Tube Exchanger
HYDROGELS FOR WOUND HEALING AND TISSUE ENGINEERING APPLICATIONS
1. Hydrogels : Applications in tissue
engineering, regenerative
medicine and wound healing
Munira Shahbuddin, PhD.
University of Sheffield, United Kingdom
International Islamic University of Malaysia, Malaysia.
Massachusetts Institute of Technology, USA.
7. Syntheticvs Natural polymers for cell
biology
Advantages
• Well defined MW
• Controlled degradation
• Simplicity – chemical structure
backbone is carbon, unlike
natural polymers which have
nitrogen, oxygen and carbon.
Disadvantages
• Synthesis and production of
polymers – derived from
petroleum
• Not sustainable
• Need cell adhesive molecules
and extracellular matrix for cell
adhesion and biological activities
8. Cells don’t like hydrogels
• Cell needs ligands for attachment.
• Cell movement is influenced by the
rigidity of substrate.
• Protein folding is influenced
almost 100% by water interactions.
Hydrophobic segments
of proteins are typically internal
segments of proteins, while
hydrophilic segments remain on
the outside, interacting
with water molecules.
Caliari. Nature Methods 13, 405–414 (2016)
10. Sample 3
- (+) GelMA
- (+) Collagen
- (+) HA
100X
HUVEC preferred to attach
on collagen surface
compared to the TCP
(competition study)
The presence of HA did not
alter/affect the morphology
of HUVEC as compared to
sample 1 and 2
(with/without HA and FN) –
Collagen is the best ECM
than FN for HUVEC on
GelMA with HA
11. Observation –
Day 2
Sample 1
(+GelMa,
+HA, -FN)
• HUVEC attached but did not spread well on
the GelMa surface when compared to the
TCP surface.
• HUVEC occupied only 15% of the surface –
observed from the cell morphology. HA
emitted autoflourescence. Need other
staining for HUVEC to distinguish from HA.
12. Schematic method for the formation of physical and chemical hydrogels
(Adapted from Advanced Drug Delivery Reviews, 54, Allan S. Hoffman,
Hydrogels for biomedical applications‘, 10, 2002).
17. Comparison of the effect of
KGM extracted from five
different species of
Amorphophallus on fibroblast
metabolic activity. (A) A.konjac
Koch, (B) A.oncophyllus, (C)
A.paeoniifolius, (D) A.praiini
and (E) A.elegans. Cell
proliferation was assessed
using MTT assay.
18. A) The stimulatory effect
of KGM on fibroblast
metabolic proliferation
mg.mL-1 extracted from 5
species, A.konjac Koch,
A.oncophyllus,
A.paeonifolius, A.prainii
and A.elegans were
dependent on
concentration of FCS.
B) Measurement of the DNA
content of fibroblasts by the
effect of KGM (A.konjac
Koch) cultured with 10% FCS
on proliferation using
PicoGreen™. DNA content of
known cell number was
calibrated with PicoGreen
19. Effect of KGM (A.konjac Koch) on human
keratinocyte proliferation assessed by MTT
assay. The bottom panel shows the viability
of cells stained with live/dead stain where
(A) are control cells and (B), (C) and (D) are
cells cultured for 5 days in the presence of
1, 5 and 10 mg.ml-1 KGM respectively
20. The relationship between the distribution of KGM (A. konjac Koch) molecular weight and ability to
stimulate fibroblasts (right) and keratinocyte proliferation and viability measured with MTT assay.
21.
22. A) B)
C) D)
(A-B, C-D) Investigation of the
ability of KGM (A. konjac Koch) to
support the metabolic activity of (A-
B) fibroblasts, (C) keratinocytes and
(D) ADMSC under conditions of
media starvation.
A) B)
C) D)
24. Blocking of MR on fibroblasts and
keratinocytes by D-mannose.
Fibroblasts Keratinocytes
25. Mechanisms of action.
Which receptors are responsible for
KGM’s biological activities on skin
cells?
Con A binds to glycosylated
surface of the cells that
consist of different type of
lectins, where semi
quantification of MR was
conducted by observation
of unbounded Con A on the
cell surface which was
blocked by D-mannose.
Photographs show dose
dependent inhibition of Con
A binding to fibroblasts. In
keratinocytes, this inhibition
was only seen at 40 mg.mL-
1
26. (A) Effect of Con A on fibroblast proliferation and (B) inhibition of the
effect of KGM on fibroblast proliferation by Con A after 1, 3 and 5 of
culture.
30. Live and dead stained (left) fibroblasts and (right) keratinocytes
with Syto9 and PI cultured in direct contact with KGM hydrogel
observed after 5 days of culture A) control, B) KGM , C) 1x10-3
D) 1.5x10-3, E) 3x10-3 and F) 6x10-3 % (w/v) of Ce (IV). 1 mL of
2x104 cells were cultured in 10% DMEM in 12 well plate. (Scale
bar: 100 μm).
34. DSC Thermograms of dried
hydrogels of (A) KGM, (B) P(NVP-
co-PEGDA), (C-E) Semi-IPN with
14, 24 and 35% (w/v) KGM,
respectively and (F-I) Graft
conetwork with 14 and 24% (w/v)
KGM and 0.5 and 1% Ce(IV)
respectively. The dash line in
purple is to show the endothermic
peak of KGM and the lines in
orange are for P(NVP-co-PEGDA)
peaks
35. Solid State 13C-NMR spectra of E)
semi IPN and F) graft-conetwork of
24% (w/v) KGM with 0.5 (blue) and
1% (w/v) Ce(IV) (black)
42. Shahbuddin et. al 2009 and Wolgemurt et. al 2011
(Images courtesy of Mark Shipman, James Blyth and
Louise Cramer, Laboratory for Molecular Cell Biology,
University College London, UK)
43. Cell-matrix interactions
• Focal adhesion is a type of
adhesive contact between the
cell and extracellular matrix
through the interaction of the
transmembrane proteins
integrins with their extracellular
ligands, and intracellular
multiprotein assemblies
connected to the actin
cytoskeleton
https://www.mechanobio.info/topics/mechanosignaling/cell-matrix-adhesion/
44. Shahbuddin et. al 2009 and Wolgemurt et. al 2011
Investigation of cell-matrix
interactions.
Cells require minimum average
distance of 30 – 50 nm spacing
between RGD islands for cell
attachment and proliferation.
45. Shahbuddin et. al 2009 and Wolgemurt et. al 2011
Faraday Discussion (July, 2008): The Importance of
Polymer Science for Biological Systems
46. Shahbuddin et. al 2014 and H.E. Huxley 1973
Keratinocytes were cultured on a graft-conetwork of 24% (w/v) KGM
with 1.0% (w/v) Ce(IV) in the presence and absence of i3T3 without
fibronectin (A and C) and with fibronectin (B and D), respectively.
47. ● represents KGM, + represents
P(NVP-co-PEGDA) while ◊
represents semi-IPN and □
represents graftconetworks
(where ■ : graft-conetworks
with 0.5% (w/v) Ce(IV) and ■ :
graft-conetworks with 1% (w/v)
Ce(IV).
48. Thermograms of hydrated (A) KGM, (B) P(NVP-co-PEGDA), (C) semi IPNs and (D) graft-conetworks
49. ● represents KGM in solution, + represents
P(NVP-co-PEGDA) while combinations of KGM
in hydrogels are shown as open symbol for
semi-IPN (, □ : 14%, ∆ : 24% and ◊ 35% (w/v)
KGM and grey symbols indicate graft-
conetwork with 0.5% Ce(IV) (where ■ ; 14 %
and ▲ : 24% (w/v) KGM) and filled symbols
indicate graft-conetwork with 1.0% Ce(IV)
(where ■ : 14% and ▲ 24% (w/v) KGM).
The relationship between free water inside the hydrogels measured by the amount of energy required
to release the water from the hydrogel measured by DSC and the water content inside the hydrogel.
50. Development of
human tissue
engineered skin model
1. Decellularizations of matrices
2. Cell culture methodologies
- Cell extraction
- Culture medium
- Maintenance
- Understanding phenotypes
3. Sterilization
4. Cell transplantation
5. Handling
51. The effects of KGM
hydrogels on human tissue
engineered skin
Figure 1. The effect of KGM and KGM
hydrogels on the reepithelisation and
stimulation of fibroblast proliferation
in the dermal region. Micrographs
show Haematoxylin and Eosin stained
TE skin cultured with 10% Green’s
medium supplemented or placed with
A) control, B) 1 mg.mL-1 KGM C) 5
mg.mL-1 KGM, D) 0.5% (w/v)
crosslinked KGM with 1x10-3% (w/v)
Ce(IV), E) 1% (w/v) crosslinked KGM
with 1x10-3% (w/v) Ce(IV) and F)
graph conetwork KGM hydrogel 24%
(w/v) with 1% (w/v) Ce(IV). Scale bar:
100 μm.
52. Figure 2. The effect of KGM and KGM hydrogels on A) the
thickness of proliferating layer of epidermal (stratum basale and
stratum granulosum), B) differentiated layer of epidermal
(startum corneum) and C) the number of cells in the dermal
region per 10,000 μm2 of TE skin after 14 days in culture with
10% Green’s supplemented with concentrations of KGM or
with either crosslinked KGM or graph conetwork KGM
hydrogels, measured using ImageJ. (n=3), ***p<0.001 highly
significant, **p<0.01 very significant and *p<0.05 significant
compared to control.
53. Figure 3. Measurement of the contraction of TE skin in direct contact with KGM, crosslinked KGM with Ce(IV)
and graft-conetwork hydrogel up until 14 days in ALI with 10% Greens’ measured using image analysis (n=3),
***p<0.001 highly significant, **p<0.01very significant and *p<0.05 significant compared to control .
54.
55.
56. Micrographs of the effect of KGM hydrogels on the
reepithelisation of a wound on TE skin with (A) control, (B) 0.5
% (w/v) crosslinked KGM, (C) 1% (w/v) crosslinked KGM and (D)
graft-conetworks hydrogel (24% (w/v) KGM and 1% (w/v)
Ce(IV) after 0, 5, 9 and 14 days in ALI with 10% Greens‘ (left)
and only on day 14 (right). Note that at C and D, the wounds
had completely healed
57. Figure 5. Measurement of the viability of wounded TE skin in ALI with 0.5 and
1 % (w/v) crosslinked KGM and graft-conetworks hydrogels after 7, 14 and 21
days in ALI with 10% Greens’ measured using Alamar Blue TM (n=2.
58.
59.
60.
61. The four rules for organ regeneration
1. In an injured organ, epithelial
layer and basal membrane
regenerate spontaneously,
stroma does not.
2. The required reactants for
inducing regeneration are
appropriate scaffold, especially
epithelial cells.
3. Scaffold are regeneratively
active if they inhibit contraction
and scar formation.
4. Structural features that
required for regenerative
activities, pore structure,
degradation rate and surface
chemistry.
Editor's Notes
Some examples where physics has shed light on biology. (a) Physics-based simulations of the walking of myosin VI predicted that the molecule would produce both hand-over-hand and inchworm type movements [8], which was later confirmed with single molecule experiments [57]. (Image courtesy of S. X. Sun, Johns Hopkins University.) (b) A crawling cell is driven primarily by the dynamics of its actin cytoskeleton, a network of filaments that polymerize at the leading edge of the cell. Force balance on the crawling cell comes from membrane forces (tension and bending), a polymerization forces, and contractile forces generated inside the cell by myosin motors and other unknown mechanisms. Here we depict a fish keratocyte, which crawls at a roughly constant speed V, while maintaining a steady cell shape. The cell body is shown in gray. (c) Stochastic simulations of microtubules determined some of the constraints for the accurate and efficient capturing of chromosomes during the formation of the mitotic spindle.
The complexity of cellular biology. (a) A subset of the chemical reactions that drive eukaryotic cell crawling. In brief, cells sense the environment through membrane bound proteins. Activation of these receptors leads to activation of a number of other proteins that promote the polymerization of actin. The biochemical reactions that govern the dynamics of actin are included. These chemical reactions produce cell motility. (b)–(d) Time series of a cancer cell (HT1080 fibrosarcoma cell) moving through a collagen I matrix. There are two hour intervals between each frame.
How physicists may be most useful for cell biology. (a) The cloud of electrons around a water molecule leads to a complex interaction potential between two water molecules (based on Ref. [58]). When we consider the bulk flow of a fluid around a ball, though, these complex interactions lead to a single bulk viscosity, which can be used to accurately calculate the fluid flows about the ball and the resistive force exerted on the ball. (b) In a similar fashion, it is possible to consider the stresses that are created inside a cell due to the dynamics of actin, without worrying about the complex reactions that create this dynamics (as depicted in Fig. 1). Combining this average dipole stress of a single cell and the adhesive interactions between neighboring cells can accurately describe the collective behavior of cells during wound healing. The bottom left panel shows the cellular flows that accompany wound healing (originally published in Ref. [56] and reprinted with permission from P. Silberzan) and a simulation from Lee and Wolgemuth (unpublished data).
Cross-bridge formation and filament sliding processes are the same in smooth muscle as they are in skeletal and cardiac muscle. Actin, myosin, and tropomyosin are all present, but smooth muscle cells do not possess troponin as their regulatory protein.
All the other muscles in the body are composed of either skeletal muscle or smooth muscle. Cardiac muscle is unique because it is striated, like skeletal muscle, yet involuntary, like smooth muscle. Skeletal muscle controls the body's movement and is generally under conscious, or voluntary, control.Oct 29, 2010