An Injectable Oxidated Hyaluronic Acid-Adipic Acid Dihydrazide Hydrogel As A Vitreous Subtitute

This article was downloaded by: [University of York]
On: 28 February 2013, At: 10:05

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954
Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Biomaterials Science,

Polymer Edition
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/tbsp20
An Injectable Oxidated Hyaluronic

Acid/Adipic Acid Dihydrazide
Hydrogel as a Vitreous Substitute
a b c
Wen-Yu Su , Ko-Hua Chen , Yu-Chun Chen , Yen-Hsien
d e f
Lee , Ching-Li Tseng & Feng-Huei Lin
a
Institute of Biomedical Engineering, National Taiwan
University, Taipei, Taiwan, R.O.C.; Division of Medical
Engineering Research, National Health Research
Institutes, 35 Keyan Road, Zhunan Town, Miaoli County
350, Taiwan, R.O.C.
b
Division of Medical Engineering Research, National
Health Research Institutes, 35 Keyan Road, Zhunan
Town, Miaoli County 350, Taiwan, R.O.C.; Department of
Ophthalmology, Taipei Veterans General Hospital, Taipei,
Taiwan, R.O.C.; National Yang-Ming University, Taipei,
Taiwan, R.O.C.
c
350, Taiwan, R.O.C.
d
Health Research Institutes, 35 Keyan Road, Zhunan Town,
Miaoli County 350, Taiwan, R.O.C.
e
Health Research Institutes, 35 Keyan Road, Zhunan Town,
Miaoli County 350, Taiwan, R.O.C.; National Science
Council, Taipei, Taiwan, R.O.C.
f
350, Taiwan, R.O.C. double@nhri.org.tw
Version of record first published: 02 Apr 2012.
To cite this article: Wen-Yu Su , Ko-Hua Chen , Yu-Chun Chen , Yen-Hsien Lee , Ching-
Li Tseng & Feng-Huei Lin (2011): An Injectable Oxidated Hyaluronic Acid/Adipic Acid
Dihydrazide Hydrogel as a Vitreous Substitute, Journal of Biomaterials Science, Polymer
Edition, 22:13, 1777-1797
To link to this article: http://dx.doi.org/10.1163/092050610X522729
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-

conditions
This article may be used for research, teaching, and private study purposes.
Any substantial or systematic reproduction, redistribution, reselling, loan, sub-
Downloaded by [University of York] at 10:05 28 February 2013
licensing, systematic supply, or distribution in any form to anyone is expressly

forbidden.
The publisher does not give any warranty express or implied or make any
representation that the contents will be complete or accurate or up to date. The
accuracy of any instructions, formulae, and drug doses should be independently
verified with primary sources. The publisher shall not be liable for any loss,
actions, claims, proceedings, demand, or costs or damages whatsoever or
howsoever caused arising directly or indirectly in connection with or arising out
of the use of this material.
Journal of Biomaterials Science 22 (2011) 1777–1797
brill.nl/jbs
An Injectable Oxidated Hyaluronic Acid/Adipic Acid

Dihydrazide Hydrogel as a Vitreous Substitute
Wen-Yu Su a,b , Ko-Hua Chen b,c,d , Yu-Chun Chen a,b , Yen-Hsien Lee b ,
Ching-Li Tseng b,e and Feng-Huei Lin a,b,∗
a
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan, R.O.C.
b
Division of Medical Engineering Research, National Health Research Institutes, 35 Keyan Road,
Zhunan Town, Miaoli County 350, Taiwan, R.O.C.

c
Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C.
d
National Yang-Ming University, Taipei, Taiwan, R.O.C.
e
National Science Council, Taipei, Taiwan, R.O.C.
Received 28 April 2010; accepted 16 July 2010
Abstract
Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the
vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor
after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with
appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid
(oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium
periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of
this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345).
The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending
on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE)
cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be
non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of
rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological
examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the
injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.
© Koninklijke Brill NV, Leiden, 2011
Keywords
Injectable hydrogel, hyaluronic acid, vitreous substitute, vitrectomy
*To whom correspondence should be addressed. Tel.: (886-37) 246-166, ext. 37100; Fax: (886-37) 586-
400; e-mail: double@nhri.org.tw
© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050610X522729

1778 W.-Y. Su et al. / Journal of Biomaterials Science 22 (2011) 1777–1797
1. Introduction
The vitreous body is a clear, transparent gelatinous substance in the vitreous cavity
of the eye that is posterior to the lens and anterior to the retina. It occupies two-thirds
of the ocular volume, with a weight of approx. 4 g and a volume of about 4 ml [1,
2]. The main components of vitreous body include water (98%), collagen fibrils,
glycosaminoglycans, hyaluronic acid (HA) and other rest solutes [3, 4]. Specific
diseases, age-related degeneration or trauma can lead to pathological changes in
the vitreous body, including HA degeneration and collagen precipitation, which
result in liquefaction of the matrix [5]. A degenerated or liquefied vitreous body
will lead to floater formation and eventually result in posterior vitreous detachment
and possible retinal detachment.
Among clinical treatments, pars plana vitrectomy (PPV) is one of the most im-
portant surgeries for treating a number of ocular-related diseases, including diabetic
retinopathy, complex retinal detachment (for example, due to trauma) and macular
hole [6–8], during PPV, the vitreous body is cut and aspirated, and then is typi-
cally replaced with a vitreous substitute, such as gas (air, perfluropropane or sulfur
hexafluoride) or silicone oil [9, 10]. Vitreous substitutes are used to fill vitreous
cavity and help reattach the retina after vitrectomy surgery. Postoperatively, a vitre-
ous substitute can keep the retina in position while the adhesion between the retina
and the retinal pigment epithelium (RPE) cells are formed [11]. Gases, which are
lighter than water, are useful for flattening a detached retina and keeping it attached
while healing occurs. However, it is frequently necessary to maintain a face-down
position following surgery for a week or more when gas is used. Since the 1960s sil-
icone oil is sometimes used instead of gases to keep retina attached postoperatively
in complicated retinal detachments [11, 12], or in patients unable to position post-
operatively (e.g., children), but long-term complications can occur if the silicone
oil is not removed later [13–15]. Besides, silicone oil also seems to be cytotoxic to
ocular tissues, such as corneal endothelial cells [16, 17].
Recently, numerous substitution materials for vitreous humor using natural,
semi-synthetic or synthetic polymer have been investigated, including poly(vinyl al-
cohol), poly(1-vinyl-2-pyrrolidone), polyacrylamide, poly(glyceryl methacrylate),
poly(methyl-2-acrylamido-2-methoxyacetate) and poly(2-hydroxyethylacrylate)
[18–22]. Chirila et al. [23] and Soman and Banerjee [24] reviewed the current
vitreous humor substitutes and revealed several criteria for the ideal vitreous sub-
stitute, including clarity, transparency, refractive index, sufficient rigidity to act
as a tamponade substitute, ability to allow metabolite transfer, non-absorbable
characteristics, hydrophilic composition and the ability to be injected through a
small-gauge needle. These criteria suggest that finding a proper material for a vit-
reous substitute is not an easy task.
We are currently developing an injectable, in situ-forming hydrogel that is com-
posed of oxidated hyaluronic acid (oxi-HA) and adipic acid dihydrazide (ADH) as a
vitreous substitute. As described previously, HA is one of the major components of
the vitreous humor. It has been used broadly in the area of biomaterials, tissue en-
W.-Y. Su et al. / Journal of Biomaterials Science 22 (2011) 1777–1797 1779
gineering and other related fields [25–27]. Glucose-based polymers contain a high
density of hydroxyl groups that make the polymers highly hydrophilic and further
chemically functionalized. HA was first used as vitreous substitute in the 1960s.
However, HA does not provide an appropriate tamponading effect on the retina
during surgery or afterwards, partly because of its low surface tension and its spe-
cific gravity. Besides, HA solutions have been shown to be not useful as long-term
vitreous substitute because of their relatively rapid elimination from the eye [28].
In order to improve the retention time of HA-based vitreous substitutes, sodium pe-
riodate (NaIO4 ) is used to oxidize HA to create aldehyde functional groups. Then,
oxi-HA can be cross-linked by ADH to form a clear, colorless and transparent oxi-
HA/ADH hydrogel.
In this work, the aldehyde functional groups of oxi-HA were characterized by
Fourier-transform infrared (FT-IR) analysis and the oxidation degree of oxi-HA

was determined using the trinitrobenzene sulfonic acid (TNBS) assay. Because the
refractive index (RI) is an essential characteristic of vitreous substitutes, the RI
of oxi-HA/ADH hydrogels with various compositions was also measured with a
refractometer. The gelation properties of oxi-HA/ADH hydrogels were evaluated
by rheological analysis at 4◦ C and 37◦ C. The elastic (G ) and viscous (G ) moduli
were recorded to determine the gelation time. In addition, in vitro degradation time,
swelling properties and cytotoxicity of oxi-HA/ADH were also investigated in this
study.
2. Materials and Methods

2.1. Materials
Hyaluronic acid (average molecular weight 3.2 × 105 ) was purchased from Q.P.
Sodium tetraborate decahydrate (borax), tert-butyl carbazate and adipic acid di-
hydrazide were purchased from Sigma-Aldrich. Diethyleneglycol, potassium bro-
mide (KBr) and sodium periodate (NaIO4 ) were purchased from RDH Chemical.
Trichloroacetic acid was purchased from JTB. Dialysis tube (MWCO 6000–8000)
was from Membrane Filtration Products. Human retina pigmented epithelium cells
(RPE cells, BCRC 60383) were supplied by the National Centre for Cell Sciences,
Taiwan. Cell-culture medium DMDM/F-12, trypsin-EDTA, fetal bovine serum and
penicillin–streptomycin were purchased from Gibco. The Quick Cell Proliferation
Assay Kit II was from BioVision. CytoTox 96® Non-Radioactive Cytotoxicity As-
say was from Promega and the Live/Dead Viability/Cytotoxicity kit for mammalian
cells was from Molecular Probes.
2.2. Preparation of Oxidated Hyaluronic Acid (oxi-HA)
HA was oxidized by sodium periodate (NaIO4 ) in an aqueous solution at room
temperature for 24 h. In a 300-ml beaker wrapped with aluminum foil, HA (2.00 g)
was dissolved in double-distilled water (200 ml), and then various concentrations
of sodium periodate solution were added gradually while stirring. The molar ratio
of NaIO4 to HA was 1:2, 1:1 and 1:0.5 to achieve various oxidation degrees (low,
middle and high oxi-HA). After 24 h of stirring, the reaction was stopped by the
addition of ethylene glycol with further stirring for 30 min. The resulting solution
was dialyzed in a dialysis tube with MWCO 6000–8000 (CelluSep T2 Tubings,
Uptima) for 3 days with double-distilled water. The water was changed at least three
times during the dialysis process. Finally, the dialyzed solution was lyophilized by a
freeze dryer (FDU-1100, Eyela) for 3 days to yield a white fluffy product, oxi-HA.
The obtained oxi-HA was manually pressed into small pellets for FT-IR analysis
(Jasco FTIR-4200 with ATR PRO450-S).
2.3. Determination of the Oxidation Degree
The oxidation degree of oxi-HA was quantified by measuring the number of alde-
hyde functional groups in oxi-HA using tert-butyl carbazate (t-BC). Carbazates are
well known to react with aldehydes to form stable carbazones in a similar manner
to hydrazone formation [29, 30]. Thus, the oxidation degree of oxi-HA could be
determined by measuring residual t-BC when excess t-BC (25 µl, 30.0 mM in 1%
aqueous trichloroacetic acid) was then reacted with the aldehyde functional groups
of oxi-HA (25 µl, 0.6% (w/v)) for 24 h. Residual t-BC was determined by adding
excess aqueous trinitrobenzene sulfonic acid (TNBS) solutions (500 µl, 6.0 mM)
and measuring the absorbance of the complex (trinitrophenyl derivative) at 340 nm
[31]. Various concentrations of aqueous t-BC solutions were used as standards to
obtain a calibration curve to identify the un-reacted carbazates in the experimental
samples.
2.4. Preparation of the oxi-HA/ADH Hydrogel
Various oxidation degrees of oxi-HA were dissolved in phosphate buffer solution
(pH 7.4) to a final concentration of 6% (w/v) at room temperature overnight. Adipic
acid dihydrazide (ADH) solutions (2, 4 and 8%, w/v) were prepared in another
phosphate buffer solution. Afterwards, 400 µl oxi-HA solution was transferred to
eppendorf and well mixed with 100 µl of 2, 4 and 8% (w/v) ADH solutions to form
oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogels.
2.5. Refractive Index of the oxi-HA/ADH Hydrogel
A refractometer (DR-A1 ATAGO, Japan) was used to measure the refractive index
(RI) of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogels. In brief, an
aliquot volume of liquid-state oxi-HA/ADH solution was moved to the refractome-
ter prism with a pipette tip. The refractometer prism was incubated at 37◦ C by an
isothermal circuiting water bath. After waiting for 10 min for gelation, the RI of
hydrogel was read from the digital screen.
2.6. Rheological Evaluation of the oxi-HA/ADH Hydrogel
A rheometer (HAAKE Rheostress 600, Thermo Fisher Scientific) with cone and
plate geometry (1-C35/2 Ti) was used to evaluate the rheological properties of
oxi-HA/ADH hydrogels at preservation temperature (4◦ C) and body temperature

(37◦ C). 4◦ C was used to evaluate the operation time for the surgeon to mix oxi-
HA/ADH solution and 37◦ C was used to evaluate the gelation time of oxi-HA/ADH
hydrogel. The oscillation time sweep mode was operated at 0.1 Hz, 10 Pa and ter-
minated after 15 min to determine the gelation time of oxi-HA/ADH hydrogels.
The elastic modulus G and the viscous modulus G were recorded and analyzed
with RheoWin 3 software.
2.7. In Vitro Degradation of the oxi-HA/ADH Hydrogel
The degradation properties of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8
hydrogels were evaluated by immersing the preformed hydrogel in PBS containing
10 000 unis lysozyme/ml. Lysozyme was selected for use in the degradation model
system because it is an ocular enzyme known to attack the polysaccharide moieties

[32]. Briefly, the oxi-HA and ADH solution were well mixed in an Eppendorf tube,
and immediately 300 µl of the oxi-HA/ADH solution was moved into a cylinder
mold and allowed to gel for 10 min to form a cylinder hydrogel with 0.7-mm di-
ameter and 0.8-mm height. The cylinder oxi-HA/ADH hydrogels were placed into
a 24-well culture plate and 3 ml PBS containing lysozyme was added to each well.
The initial hydrogel dry weight (Wid ) was determined immediately after the hydro-
gel formed. At regular intervals, the hydrogels were removed and lyophilized by
freeze-drier for 72 h. The dry weight (Wd ) of oxi-HA/ADH hydrogels at different
time point was determined and the degradation percentage was calculated using the
equation:
Degradation (%) = ((Wid − Wd )/Wid ). (1)
2.8. Swelling of the oxi-HA/ADH Hydrogel
The swelling index of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydro-
gels was evaluated under the same conditions as the in vitro degradation exper-
iments. The initial hydrogel weight (Wi ) was determined immediately after the
hydrogel was formed and then placed into a 24-well culture plate. At regular in-
tervals, hydrogels were removed from the PBS containing lysozyme, blotted with
filter paper to remove surface water, weighed (Wt ) and returned to the same con-
tainer (the buffer solution was replaced at each measurement). The swelling index
was calculated from the ratio between Wt and Wi .
2.9. Cytotoxicity of the oxi-HA/ADH Hydrogel
The cytotoxicty of the oxi-HA/ADH hydrogel was determined by testing the ex-
traction medium with a monolayer of human retina pigmented epithelium cells
(RPE cells, BCRC 60383, National Centre for Cell Sciences, Taiwan) according
to ISO standards [33]. The extraction medium was prepared by incubating the
oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogels with standard culture
medium (DMEM/F-12) at a 0.75 cm2 /ml extraction ratio for 72 h at 37◦ C. Extrac-
tion medium (200 µl) was tested on a monolayer of RPE cells. RPE cells were
seeded in 96-well culture plates at a cell density of 5 × 103 cells/well and fed with
standard culture medium at 37◦ C overnight. The standard culture medium was then
replaced with the extraction medium. Groups in the experiment including control
(standard culture medium), negative control (Al2 O3 extraction medium), positive
control (0.1% Triton X-100 containing medium) and experimental groups (oxi-
HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 extraction medium) were tested in
hexplicate (n = 6). After incubation at 37◦ C for 1 and 3 days, cell viability and
cytotoxicity evaluations were quantitatively assessed using the Quick Cell Prolifer-
ation Assay Kit II (BioVision) and the CytoTox 96® Non-Radioactive Cytotoxicity
Assay (Promega), separately. RPE cells treated with extraction medium were also
stained with the Live/Dead staining kit (Molecular Probes, Cat. No. L3224).
The Quick Cell Proliferation Assay Kit II was used to evaluate the cell viability.
After cultured for 1 and 3 days, the medium was discarded and replaced with 0.2 ml
water-soluble tetrazolium-8 (WST-8) working solution to each well. WST-8 can be
reduced by dehydrogenases in living cells to produce a yellow colored product (for-
mazan). After 2 h incubation, 100 µl working solution was quantitatively assessed
by a spectrophotometer readout at 450 nm. The reference wavelength was 650 nm.
The CytoTox® 96 Non-Radioactive Cytotoxicity Assay kit was used to evalu-
ate the cytotoxicity. The assay kit quantitatively measures lactate dehydrogenase
(LDH), which is a stable cytosolic enzyme released upon cell lysis. After 1 and
3 days cultivation, both culture medium and cell total lysis were measured by the
absorbance at 490 nm according to the assay manual. Extraction medium (without
incubation with RPE cells) was also evaluated to serve as background. The cyto-
toxicity was calculated by the following equation:

ODculture medium − ODbackground
Cytotoxicity (%) = × 100%.
ODtotal lysis + ODculture medium − ODbackground
2.10. Preliminary Animal Study
Six eyes of three New Zealand white rabbits (2.8–3.2 kg) were used. The surg-
eries were performed under general anesthesia with intramuscular injection of
ketalar/Chanazine 2%. Under an operating microscope, using a surgical blade, a
sclerotomy was created approx. 3 mm in the left eyes. The vitreous body was
aspirated by 18-gauge needles as much as possible and replaced with air. Then oxi-
HA/ADH solution was injected into the vitreous cavity. After surgery, the eyes were
treated with gentamicin (genticin, Roche) as antibiotic eyedrops and tetracyclin hy-
drochloride ophthalmic ointment 3 times a day for 1 week. The right eyes were
used as control eyes without any surgery. An ophthalmic table slit lamp (Topcon
Medical Systems) was used to observe and record the anterior segment and ocular
media. Intraocular pressure (IOP) was measured using a Schiotz tonometer at 1,
5, 8, 12, 15 and 3 weeks postoperation. Ultrasonic pachymetry (DGH Technology)
was used to measure the central cornea thinkness. Three weeks after operation, the
rabbits were killed. Both the right (control) and left (operated) eyes were harvested
from these animals. The eyes were fixated with 10% formaldehyde solution and
they were embedded in paraffin and stained with hematoxylin and eosin (HE stain)
for a light-microscopic observation.
2.11. Statistical Analysis
All data are expressed as mean ± SD. Statistical differences between groups were
tested using one-way analysis of variance (ANOVA). Statistical significance was
set in advance to a probability level of 0.05.
3. Results
3.1. Characterization of oxi-HA
In the study, HA was oxidated by different concentrations of NaIO4 at room tem-

perature. Figure 1a shows the chemical reaction of HA oxidated by NaIO4 to create
two aldehyde functional groups. The FT-IR spectra of HA powder and various oxi-
dation degrees of oxi-HA (low, middle and high oxi-HA) are shown in Fig. 1b. The
significant peaks of the aldehyde functional group could be observed in the FT-IR
spectra (ii, iii and iv) at 1725 and 863 cm−1 , and the peak intensity increased as the
oxidation degree increased. The peaks at 1147 and 895 cm−1 (i), which are related
to C–O–C (ether bond) and C–H, shifted to 1112 and 875 cm−1 (ii, iii and iv) due
to the formation of aldehyde functional groups.
The oxidation degree of oxi-HA was further measured by t-BC titration (TNBS
assay) as described previously. Table 1 summarizes the theoretical oxidation degree
that was calculated from the molar ratio of NaIO4 and HA, and the obtained ox-
idation degree that was measured by the TNBS assay. As expected, the oxidation
degree of oxi-HA was increased from 27.3 ± 2.3% to 60.4 ± 2.66% as the amount
of NaIO4 increased. The oxi-HA with middle oxidation degree (44.3 ± 4.25%) was
cross-linked with various concentration of ADH (2, 4 and 8%, w/v) for the follow-
ing experiments.
3.2. Refractive Index (RI) of the oxi-HA/ADH Hydrogel
Oxi-HA could be cross-linked with the ADH, a bi-functional cross-linker, to form
hydrogels without the use of any chemical initiator (Fig. 2a). Aldehyde functional
groups of oxi-HA can react with the NH2 functional group of ADH very rapidly
to form a colorless, transparent oxi-HA/ADH hydrogel (Fig. 2b). The RI of the
oxi-HA/ADH hydrogels ranged from 1.3420 to 1.3442 as the ADH concentration
increased from 2 to 8% (Fig. 2c), very close to that of human vitreous humor
(1.3345–1.3348) [34].
3.3. Rheological Properties of the oxi-HA/ADH Hydrogel
Oscillatory time sweeps were performed to evaluate the gelation behavior of the
oxi-HA/ADH hydrogel. Figure 3 shows the elastic modulus (G ) and viscous mod-
ulus (G ) of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogel at 4◦ C
and 37◦ C. The cross-over point of G and G was defined as gel point, indicating
(a)
(b)
Figure 1. (a) The chemical reaction of hyaluronic acid (HA) oxidized by NaIO4 ; (b) FT-IR spectrum
of oxidated hyaluronic acid (oxi-HA) with different oxidation degree: (i) HA powder (oxidation degree
0%), (ii) low oxi-HA (oxidation degree 27.3%), (iii) middle oxi-HA (oxidation degree 44.3%) and
(iv) high oxi-HA (oxidation degree 60.4%). The arrow indicates the aldehyde functional groups of
oxi-HA at 1725 and 836 cm−1 and the shifting peaks of the C–O–C and C–H bands at 1147 and
895 cm−1 . This figure is published in colour in the online edition of this journal, that can be accessed
via http://www.brill.nl/jbs
gel formation. The time required for the gel point to occur is sometimes referred to
as the gelation time for the samples [35, 36].
Figure 3a shows the rheological results of oxi-HA/ADH hydrogels (oxi-
HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8) at 4◦ C. These results indicate
that all types of oxi-HA/ADH hydrogels can maintain the liquid state at 4◦ C for
Table 1.
Oxidation degree and yield percentage of HA oxidated by NaIO4 for 24 h
Low oxi-HA Middle oxi-HA High oxi-HA
HA/NaIO4 molar ratio 1:0.5 1:1 1:2

Theoretical oxidation degree 50 100 100
Oxidation degree (%) 27.30 ± 2.36 44.33 ± 4.25 60.40 ± 2.66
Yield (%) 87.62 ± 2.94 84.04 ± 5.75 87.53 ± 4.42
The oxidation degree was measured by TNBS assay.
3–8 min, depending on the concentration of ADH in the hydrogel. Figure 3b shows
the rheological results of oxi-HA/ADH hydrogels at 37◦ C. The gel point of all
hydrogels appears between 143 and 175 s, which means that the oxi-HA/ADH hy-
drogels begin to transform into a gel matrix within 3 min at 37◦ C. The rheological
evaluation results of oxi-HA/ADH hydrogels are summarized in Table 2.
3.4. In Vitro Degradation and Swelling Index of the oxi-HA/ADH Hydrogel
Figure 4a shows the mass remaining percentage of oxi-HA/ADH hydrogel as
a function of time. The oxi-HA/ADH2 and oxi-HA/ADH4 hydrogel dissolved
completely on day 3 and day 14, respectively. On day 3, the mass remaining
percentage of oxi-HA/ADH8 hydrogel was 86.67 ± 3.54%, gradually decreas-
ing to 61.02 ± 3.13% on day 35. Figure 4b shows the swelling index of oxi-HA
cross-linked with different ADH concentrations. For oxi-HA/ADH2 hydrogel, the
swelling index increases over time until the hydrogel was completely dissolved
(within 3 days). For oxi-HA/ADH4 and oxi-HA/ADH8, the swelling index slightly
decreases during the first 3 days and then maintains a constant value until the hy-
drogels begin to degrade.
3.5. Cytotoxicity of the oxi-HA/ADH Hydrogel
Cell viability was evaluated by WST-8 assay (Fig. 5a). There was no significant
difference among the experimental groups (oxi-HA/ADH2, oxi-HA/ADH4 and oxi-
HA/ADH8) on the first day (P > 0.5). After culturing for 3 days, the WST-8 OD450
values of the control groups, oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8
were 1.27±0.03, 1.20±0.05, 1.14±0.04 and 0.99±0.06, respectively. The extrac-
tion medium of oxi-HA/ADH2 and oxi-HA/ADH4 did not significantly influence
the cell viability of RPE cell compared to the control and negative control group
(P > 0.5). However, the extraction medium of oxi-HA/ADH8 causes a small reduc-
tion in cell viability compared to the control group (P < 0.001). We further checked
the cytotoxicity by LDH assay (Fig. 5b); the cytotoxicity of oxi-HA/ADH2, oxi-
HA/ADH4 and oxi-HA/ADH8 was 6.1640 ± 0.5805%, 6.0720 ± 0.3872% and
5.3166 ± 0.9590%, respectively. Results showed that there was no significant dif-
ference among all oxi-HA/ADH extraction medium groups compared to the control
group (6.9811 ± 0.6663%, P > 0.5).
(a)
(b)
(c)
Figure 2. (a) Scheme of covalent cross-linking of oxi-HA with ADH. (b) Oxi-HA/ADH solution can
be injected through a 27-gauge needle and form the colorless and transparent hydrogel. (c) Refractive
index of 6% (w/v) oxi-HA (oxidation degree 44.3%) cross-linked with various concentrations of ADH
(2–8%, w/v). This figure is published in colour in the online edition of this journal, that can be accessed
via http://www.brill.nl/jbs
(a)
Figure 3. Rheological evaluation of 6% (w/v) oxi-HA (oxidation degree of 44.3%) cross-linked with
various concentrations of ADH (2, 4 and 8%, w/v) to form oxi-HA/ADH hydrogel at (a) 4◦ C and
(b) 37◦ C. Elastic modulus (G , !) and viscous modulus (G , ") of the oxi-HA/ADH hydrogel were
measured at a constant frequency of 0.1 Hz as a function of time. The gel point is defined as the
cross-over point of G and G . The time required for the gel point to occur is referred to as the
gelation time.
Figure 6 shows fluorescence photomicrographs of the RPE cells cultured in the

oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogel extraction medium.
The polyanionic dye calcein-AM is well retained within live cells and produces
an intense uniform green fluorescence in live cells. On the other hand, EthD-1 en-
ters cells with damaged membranes and produces a bright red fluorescence in dead
cells. Live/dead staining demonstrates that most of the cells cultured in different
extraction media were viable.
3.6. Preliminary Animal Study
Figure 7a shows the changes of the intraocular pressure (IOP) after operation. The
IOP of operated eyes slightly decreased on postoperative day 1. There were upward
trends of IOP at 5 and 8 days after the injection of oxi-HA/ADH hydrogel. At 12, 15
and 21 days, the IOP reached a plateau level. The result showed no significant dif-
ference between the operated eyes and control eyes during the observation period.
Figure 7b shows the cornea thickness of operated eyes and control eyes, and there
were no significant differences in each group. Figure 8a shows a photograph of slit-
(b)
Figure 3. (Continued.)
Table 2.
Characteristics of oxi-HA/ADH hydrogels prepared with 6% (w/v) oxi-HA cross-linked with different
concentrations of ADH (2, 4 and 8%, w/v)
Hydrogel RI Gel point (s) In vitro degradation

time (days)
4◦ C 37◦ C
Oxi-HA/ADH2 1.3420 ± 0.0000 180.3 175.4 2

Oxi-HA/ADH4 1.3427 ± 0.0001 202.2 185.7 14
Oxi-HA/ADH8 1.3442 ± 0.0001 491.7 143.4 >35
The gel point was defined at the cross-over point of G and G determined by rheological mea-
surement (G = G ); the time required for the gel point to occur is referred to as the gelation time.
lamp examinations at 21 days after the injection of oxi-HA/ADH8. The cornea and
lens of the operated eye presented no defect. These results revealed no significant
inflammation or other disease of the anterior segment in examinations at 3 weeks
postoperation. Figure 8b shows the retina sections of operated and control eyes at
21 days. The retinal layers were easily detected, and there was no inflammatory
reaction or infiltration, and also no differences from the control eyes.
(a)
(b)
Figure 4. In vitro degradation evaluation of oxi-HA/ADH hydrogel. (a) Mass remaining percentage
and (b) swelling index of oxi-HA/ADH2 (E), oxi-HA/ADH4 (P) and oxi-HA/ADH8 (1). The cylin-
der hydrogels (300 µl) were immersed in 3 ml PBS containing 10 000 U lysozyme/ml.
(a)
(b)
Figure 5. Cytotoxicity results of (a) optical density readings obtained in the WST-8 assay related to
the cell proliferation (n = 6) and (b) cytotoxicity evaluation of the cells cultured in extraction medium
of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8 hydrogel (n = 6) at 1 and 3 days.
4. Discussion
When a patient undergoes vitrectomy surgery, much of the vitreous humor is re-
moved from the vitreous cavity. Because the natural vitreous cavity is unable to
regenerate, the vitreous cavity must be filled with vitreous substitute to keep the
retina in position and prevent it from detaching again. In past decades, the de-
Figure 6. Fluorescent photomicrographs (40×, scale bars 200 µm) of retina pigmented epithelium
(RPE) cells cultured in DMEM/F-12 medium (control), DMEM-F12 medium containing 0.1% Triton
X-100 (positive control) and extraction medium of oxi-HA/ADH2, oxi-HA/ADH4 and oxi-HA/ADH8
hydrogel at 1 and 3 days. The polyanionic dye calcein-AM is well retained within live cells, producing
an intense uniform green fluorescence in live cells. This figure is published in colour in the online
edition of this journal, that can be accessed via http://www.brill.nl/jbs
(a)
(b)
Figure 7. (a) Intraocular changes. No significant elevation of intraocular pressure was observed during
the observation period, and no significant differences were observed between the operated eyes and
the control eyes. (b) Changes in cornea thickness. No significant changes of cornea thickness were
observed throughout the observation period, and no significant differences were observed between the
operated eyes and the control eyes (n = 3).
velopment of vitreous substitutes has been challenging for researchers, and many
biopolymers and synthetic compounds have been examined in laboratories. How-
(a)
(b)
Figure 8. (a) Slit-lamp photograph of the oxi-HA/ADH8 injected eye 3 weeks postoperatively and
control eye. There was no significant between operated eye (left) and control eye (right), the cornea
was clear, and there was no evidence of clinically significant anterior chamber inflammation. (b) Light
microscope observation of retina histological section at day 21 after surgery (HE stain, 200×). No sig-
nificant difference was observed between the operated eye (left) and control eye (right). This figure is
published in colour in the online edition of this journal, that can be accessed via http://www.brill.nl/jbs
ever, the ideal material for permanent vitreous substitute has not been developed at
present.
This study proposed a colorless and transparent oxi-HA/ADH hydrogel that can
be injected into the vitreous cavity through a 27-gauge needle and subsequently
transforms into a gel-like substance. The aldehyde functional groups on oxi-HA
were created by NaIO4 , which is a well-known oxidizing agent, cleaving the C2-
C3 hydroxyl groups to form dialdehyde which was characterized by FT-IR (peaks
at 1725 and 863 cm−1 ), as shown in Fig. 1b. The dialdehyde of oxi-HA could react
with the hydrazide group of ADH to form intermolecular networks in oxi-HA/ADH

hydrogel.
The RI is one of the important optical characteristics for the application of vit-
reous substitutes. An inappropriate RI index of a vitreous substitute will influence
the eyesight of a patient after vitrectomy. Gases tamponade induce optical changes
that are severe enough to limit temporally both the funduscopic examination and
patient’s eyesight [39]. Due to the RI values of current vitreous substitutes (e.g.,
silicone oil, RI = 1.40516; heavy silicone oil, RI = 1.3008 [24]) differ from that of
the natural vitreous body (RI = 1.336), induced refractive shifts in tamponade eyes
are expected. The RI of oxi-HA/ADH8 is about 1.3342, which is quite similar to
that of the human vitreous body. The use of oxi-HA/ADH8 hydrogel as a vitreous
substitute can avoid the undesired refractive changes after vitrectomy.
Clinically, injection through a small-gauge needle is the common procedure for

the delivery of a vitreous substitute. The oxi-HA/ADH8 hydrogel can be easily in-
jected through a 27-gauge needle, as Fig. 2b shows. The injectable ability is an
important factor to ocular surgeons and patients. The operation time should be suf-
ficient for the ocular surgeon to inject the liquid-state hydrogel into the vitreous
cavity, and the time of solution–gel transformation should be as short as possible to
prevent extrusion of the hydrogel. The rheological evaluation showed that a mix-
ture of oxi-HA solution and ADH solution could maintain in solution form for about
8 min. According to these results, the ocular surgeons have sufficient time (8 min)
to mix oxi-HA and ADH solution at 4◦ C, and then transfer the mixed oxi-HA/ADH
solution into a syringe for injection. It overcomes the difficulties of introducing the
substitute in the gel form, and can lead to a minimally invasive delivery system for
the vitreous substitute to the eye by injection of the liquid.
Some studies suggested that the reaction between aldehydes and hydrazides is
very fast, and the resulting hydrazone bonds will be labile to hydrolysis [40]. For
low cross-linked hydrogel (oxi-HA/ADH2), the swelling index increased immedi-
ately and dissolved completely within 3 days. During this time, the hydrogel under-
goes hydrolysis and the hydrazone bonds are degraded almost linearly with time.
For high cross-linked hydrogels (oxi-HA/ADH4 and oxi-HA/ADH8), the swelling
index decreased during the first 3 days followed by a constant value and then in-
creased until the networks were completely dissolved. The increase of swelling
index during time is a consequence of the hydrolysis of the hydrazone bonds in
the hydrogel network. When hydrazone bonds in the hydrogel are hydrolyzed, the
network swells and contains more water, and then dissolves completely. Accord-
ing to the mass loss studies, the degradation behavior found in oxi-HA/ADH4 and
oxi-HA/ADH8 hydrogels is different from the one observed in oxi-HA/ADH2 hy-
drogels. Hydrogel with higher ADH content and, therefore, with a higher number of
hydrazone bonds, tend to hydrolyze slower than that with lower ADH content. In the
high cross-linked hydrogels, there is an initial decrease in the mass loss (5–15%)
for the first 3 days, likely due to the hydrolysis of the hydrogel regions with low
cross-linking density or un-cross-linked HA. This phase was followed by a slight
decrease in the mass during the next few days or weeks, likely due to the degrada-
tion of high cross-linked regions. Finally, the hydrazone bonds in the hydrogel were
hydrolyzed, resulting in a dissolution phase.
The oxi-HA/ADH4 hydrogel showed a progressive swelling at 8–12 days, which
means the hydrazone bonds were hydrolyzed at this time-point, and then followed
by a dissolution phase at 14 days. This results in a decrease in hydrogel mass and
finally in a complete dissolution of the hydrogel. On the other hand, due to the high
cross-linking degree of the hydrogel, oxi-HA/ADH8 reached a constant swelling
index at 3 days, and the hydrogel showed a progressive swelling at 30–35 days.
The in vitro degradation experiments showed that only 30–40% of oxi-HA/ADH8
hydrogel was degraded in the presence of enzymes (PBS containing 10 000 units
lysozyme/ml), and the hydrogel was stable for 5 weeks. Clinically, silicone oil
and gases are the common vitreous substitutes in retina reattachment surgery to re-
establish the intraocular volume and manipulate retina detachments [39, 40]. They
prevent passage of fluid through retina breaks, maintain normal retina–retina pig-
ment epithelium (RPE) apposition and maintain retinal reattachment. The longevity
of gases is usually within few days to 2 weeks, depending on the gas type, bubble
volume, initial concentration and intraocular pressure [39, 41]. In contrast to in-
traocular gases, which reabsorb within few weeks, the oxi-HA/ADH8 hydrogel can
maintain the gel matrix for at least 5 weeks in the in vitro degradation test. For clin-
ical consideration, the longevity of oxi-HA/ADH8 hydrogel should be beneficial
for retina attachment. Furthermore, the hydrogel overcomes the inconvenience of
face-down position when patients undergo the vitrectomy with gases tamponade.
Retina pigmented epithelium (REP) cells were selected as target cells for cyto-
toxicty evaluation because they are often directly exposed to the vitreous substitute
filling the globe after retina detachment or local retinectomy [42, 43]. RPE cell
is also associated with failed retinal reattachment and proliferative vitreoretinopa-
thy (PVR) according to Machemer and LaQua [44] and Glaser et al. [45]. The
cell viability (WST-8) and cytotoxicity (LDH assay) assays indicate that the oxi-
HA/ADH hydrogel was biocompatible and non-toxic with RPE cells in in vitro
evaluation. Degradation products of hydrogel are another concern for clinical ap-
plication. According to the results of degradation evaluation, the oxi-HA/ADH2
hydrogel was degraded within 3 days in aqueous environments. This means that
oxi-HA/ADH2 hydrogel totally degraded in the extraction medium during extrac-
tion process (37◦ C, 72 h) and the degraded products were culture with RPE cell
in cytotoxicity evaluation. According to the cytotoxicity assay data (Fig. 5b), there
was no significant cytotoxicity in this group. These results indicated that the degra-
dation products from the oxi-HA/ADH hydrogel should be non-cytotoxic to RPE
cells based on the WST-8 and cytotoxicity assay. In addition, Live/Dead staining
also demonstrates that most of the cultured cells from different extraction media
were viable.
In oxi-HA/ADH8 injected eyes, slit-lamp examination, postoperative intraocu-
lar pressure and cornea thickness revealed no findings of inflammation or opacity
in the anterior ocular segment during observation period. The increasing IOP of-
ten occurs after vitrectomy, but the IOP of operated eyes remains at normal level
during the observation period. This may be due to a slightly decrease in swelling
index of oxi-HA/ADH hydrogel. Histological examination of the retina tissue also
showed no structure changes or degradation, and also no significant difference form
the control eyes. Although the oxi-HA/ADH injection did not induce abnormal bi-
ological reactions, the function of the retina in operated eyes was still unknown.
More studies are required to evaluate the function of the eye.
5. Conclusion
Currently, ideal vitreous substitutes have not been developed yet for clinical appli-
cation. Many researchers made efforts toward developing appropriate biomaterials
for vitreous substitutes. In this study, we report the synthesis and characterization of
a novel injectable HA-based hydrogel, oxi-HA/ADH. The hydrogel displays several
beneficial properties for the application of vitreous substitute, such as appropriate
RI, injectable and in situ gelling properties, and cytotoxicty with REP cells. Addi-
tionally, the oxi-HA/ADH8 hydrogel was not degraded in vitro by ocular enzyme
(lysozyme) over 5 weeks. In the preliminary animal study, the oxi-HA/ADH hydro-
gel induced no serious complication during the observation period; however, further
studies are required to examine the function of retina before the oxi-HA/ADH hy-
drogel can be used clinically.
References
1. D. A. Swann and I. J. Constable, Invest. Ophthalmol. Vis. Sci. 11, 159 (1972).
2. D. A. Swann and I. J. Constable, Invest. Ophthalmol. Vis. Sci. 11, 164 (1972).
3. J. V. Forrester, A. D. Dick, P. McMenamin and W. R. Lee, in: The Eye: Basic Science in Practice,
W. B. Saunders, p. 182. London (1996).
4. S. Nishikawa and T. Makoto, Curr. Eye Res. 15, 37 (1996).
5. L. I. Los, R. J. van der Worp, M. J. A. van Luyn and J. M. M. Hooymans, Invest. Ophthalmol. Vis.
Sci. 44, 2828 (2003).
6. V. Lakhanpal, S. S. Schocket, M. J. Elman and M. R. Dogra, Ophthalmology 97, 1114 (1990).
7. F. M. Recchia, A. J. Ruby and C. A. Carvalho Recchia, Am. J. Ophthalmol. 139, 447 (2005).
8. T. G. Sheidow and J. R. Gonder, Retina 18, 510 (1998).
9. W. J. Foster, Expert Rev. Ophthalmol. 3, 211 (2008).
10. K. E. Swindle and N. Ravi, Expert Rev. Ophthalmol. 2, 255 (2007).
11. G. G. Giordano and M. F. Refojo, Prog. Polym. Sci. 23, 509 (1998).
12. P. A. Cibis, B. Becker, E. Okun and S. Canaan, Arch. Ophthalmol. 68, 590 (1962).
13. D. J. Light, Optometry 77, 446 (2006).
14. T. Krupin, A. E. Kolker and L. F. Rosenberg, in: Complications in Ophthalmic Surgery, p. 329.
Mosby, Dublin, OH (1999).
15. A. Crisp, E. de Juan and J. Tiedeman, Arch. Ophthalmol. 105, 546 (1987).
16. C. S. Yang, K. H. Chen, W. M. Hsu and Y. S. Li, Eye 22, 282 (2008).
17. K. Nakamura, M. F. Refojo, D. V. Crabtree, J. Pastor and F. L. Leong, Invest. Ophthalmol. Vis.
Sci. 32, 3007 (1991).
18. Y. Hong, T. V. Chirila, S. Vijayasekaran, W. Shen, X. Lou and P. D. Dalton, J. Biomed. Mater. Res.
39, 650 (1998).
19. S. Maruoka, T. Matsuura, K. Kawasaki, M. Okamoto, H. Yoshiaki, M. Kodama, M. Sugiyama and
M. Annaka, Curr. Eye Res. 31, 599 (2006).
20. S. Suri and R. Banerjee, J. Biomed. Mater. Res. A 79, 650 (2006).
21. K. E. Swindle, P. D. Hamilton and N. Ravi, Polym. Prepr. 47, 59 (2006).
22. K. E. Swindle, P. D. Hamilton and N. Ravi, J. Biomed. Mater. Res. A 78, 656 (2008).
23. T. V. Chirila, S. Tahija, Y. Hong, S. Vijayasekaran and I. J. Constable, J. Biomater. Appl. 9, 121
(1994).
24. N. Soman and R. Banerjee, Biomed. Mater. Eng. 13, 59 (2003).
25. N. J. Turner, C. M. Kielty, M. G. Walker and A. E. Canfield, Biomaterials 25, 5955 (2004).
26. H. S. Yoo, E. A. Lee, J. I. Yoon and T. G. Park, Biomaterials 26, 1925 (2005).
27. D. D. Allison and K. J. Grande-Allen, Tissue Eng. 12, 2131 (2006).
28. T. V. Chirila, Y. Hong, P. D. Dalton, I. J. Constable and M. F. Refojo, Prog. Polym. Sci. 23, 475
(1998).
29. K. H. Bouhadir, D. S. Hausman and D. J. Mooney, Polymer 40, 3575 (1999).
30. X. Jia, J. A. Burdick, J. Kobler, R. J. Clifton, J. J. Rosowski, S. M. Zeitels and R. Langer, Macro-
molecules 37, 3239 (2004).
31. K. Y. Lee, K. H. Bouhadir and D. J. Mooney, Macromolecules 33, 97 (1999).
32. I. G. Rennie and M. A. Parsons, Arch. Ophthalmol. 99, 1850 (1981).
33. International Standardization Organization, Biological Evaluation of Medical Devices. Part 5.
Tests for Cytotoxicity: in vitro Methods, ISO10993-5 (1992).
34. W. S. Duke-Elder, J. Physiol. 68, 155 (1929).
35. W. B. Yoon, B. Y. Kim and J. W. Park, J. Food Sci. 64, 291 (1999).
36. S. Mortimer, A. J. Ryan and J. L. Stanford, Macromolecules 34, 2973 (2001).
37. J. D. Zhang, M. J. Yang, Y. R. Zhu and H. Yang, Polym. Int. 55, 951 (2006).
38. J. Maia, L. Ferreira, R. Carvalho, M. A. Ramos and M. H. Gil, Polymer 46, 9604 (2005).
39. M. G. Krzystolik and D. J. D’Amico, Int. Ophthalmol. Clin. 40, 187 (2000).
40. B. Balakrishnan, M. Mohanty, P. R. Umashankar and A. Jayakrishnan, Biomaterials 26, 6335
(2005).
41. I. J. Constable and D. A. Swann, Arch. Ophthalmol. 93, 416 (1975).
42. T. R. Friberg, T. C. Verstraeten and D. K. Wilcox, Invest. Ophthalmol. Vis. Sci. 32, 2030 (1991).
43. J. Fernandez-Vigo, M. F. Refojo and T. Verstraeten, Retina 10, 148 (1990).
44. R. Machemer and H. LaQua, Am. J. Ophthalmol. 80, 1 (1975).
45. B. M. Glaser, A. Cardin and B. Biscoe, Ophthalmology 94, 327 (1987).

An Injectable Oxidated Hyaluronic Acid-Adipic Acid Dihydrazide Hydrogel As A Vitreous Subtitute

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

An Injectable Oxidated Hyaluronic Acid-Adipic Acid Dihydrazide Hydrogel As A Vitreous Subtitute

Uploaded by

Copyright:

Available Formats

This article was downloaded by: [University of York]

On: 28 February 2013, At: 10:05

Journal of Biomaterials Science,

An Injectable Oxidated Hyaluronic

To link to this article: http://dx.doi.org/10.1163/092050610X522729

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-

licensing, systematic supply, or distribution in any form to anyone is expressly

An Injectable Oxidated Hyaluronic Acid/Adipic Acid

Zhunan Town, Miaoli County 350, Taiwan, R.O.C.

Received 28 April 2010; accepted 16 July 2010

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050610X522729

Fourier-transform infrared (FT-IR) analysis and the oxidation degree of oxi-HA

2. Materials and Methods

oxi-HA/ADH hydrogels at preservation temperature (4◦ C) and body temperature

system because it is an ocular enzyme known to attack the polysaccharide moieties

In the study, HA was oxidated by different concentrations of NaIO4 at room tem-

Low oxi-HA Middle oxi-HA High oxi-HA

HA/NaIO4 molar ratio 1:0.5 1:1 1:2

The oxidation degree was measured by TNBS assay.

Figure 6 shows fluorescence photomicrographs of the RPE cells cultured in the

Hydrogel RI Gel point (s) In vitro degradation

Oxi-HA/ADH2 1.3420 ± 0.0000 180.3 175.4 2

with the hydrazide group of ADH to form intermolecular networks in oxi-HA/ADH

Clinically, injection through a small-gauge needle is the common procedure for

You might also like