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Journal of Medical and Biological Engineering, 29(6): 304-310 304
Guided Tissue Regeneration with Use of CaSO4-Chitosan
Composite Membrane
Shyh-Ming Kuo Gregory Cheng-Chie Niu Cheng-Wen Lan Min-Feng Cheng
Min-Yu Chiang Shwu-Jen Chang*
Department of Biomedical Engineering, I-Shou University, Kaohsiung 840, Taiwan, ROC
Received 7 Jul 2009; Accepted 10 Oct 2009
Abstract
Guided tissue regeneration (GTR) membranes with bioresorbable characteristics have been employed in recent
years for periodontal procedures to deflect the growth of gingival tissues away from root surface. They provide an
isolated space over regions with defective tissues and allow the relatively slow-growing periodontal ligament
fibroblasts to be repopulated over the root surface. In this study, we employed chitosan and CaSO4 composites as one
of the viable membrane materials and evaluated their roles in GTR applications. Two types of CaSO4-chitosan
composite membranes with molecular weights of chitosan 70 kDa and 300 kDa, respectively, were prepared for study
in three categories: mechanical strength to create and sustain an effective space; rapid rate to reach hydrolytic
equilibrium in phosphate buffer solution; and ease of clinical manipulations. Consequently, standardized, trans-osseous
and critical-sized (cavity of 6 mm) skull defects were made in adult rats, and the defective regions were covered with
the specifically prepared chitosan membranes. After 4 weeks of recovering, varying degrees of bone healing were
observed beneath the CaSO4-chitosan composite membranes in comparison to the control group. The CaSO4-chitosan
composite membrane-covered regions showed a clear boundary space between connective tissues and bony tissues. In
summary, good cell-occlusion and beneficial osteogensis effects by these bioresorbable GTR materials toward the
wound recovery were indicated.
Keywords: Guided tissue regeneration (GTR), Chitosan, CaSO4, Membrane
1. Introduction
Guided tissue regeneration (GTR) techniques have been
successfully applied in the treatment of periodontal diseases
and provided opportunity for formation of new bone [1-3]. The
techniques involved procedures employing membranes as
mechanical barriers to create a space around the defects that
might permit bone regeneration and to prevent the epithelial
cells from migrating into the bony area. In general, an effective
barrier membrane should have appropriate mechanical strength
to occlude the rapid growth of repairing tissues (epithelium and
gingival connective tissue) away from the root surface and
maintain a protective space over the defect that will allow
migration of bone cells from the surrounding alveolar bone into
the targeted area. Ideally, clinical manageability of the barrier
membrane is also a necessary consideration during surgical
procedures. In addition, effective materials should be safe,
non-toxic, and non-antigenic ones which induce little or no
* Corresponding author: Shwu Jen Chang
Tel: +886-7-6577711 ext. 6719; Fax: +886-7-6577067
E-mail: [email protected]
inflammatory responses around the host tissue. Furthermore,
the barrier function must be established and maintained for a
period of time long enough for tissue guidance to take effect.
As a result, extensive effort has been employed to utilize
bio-resorbable membranes to achieve therapeutic purpose in
clinical trials [4-7].
Among the bio-resorbable materials used in GTR
applications, resorbable membranes such as collagen
membranes and polyglycolic acid-based membranes, have been
successfully applied and are commercially available. However,
there could be disadvantages in the use of collagen membrane
because it might cause localized chronic inflammatory response
and rapid degradation behavior [8,9]. As for the use of
polyglycolic acid-based membrane, there could be problems
such as gingival recession, exposure of the device, and
inflammation of surrounding soft tissue [10,11].
Chitosan, a natural occurring polysaccharide, is widely
present among marine and terrestrial invertebrates and lower
forms of the plant kingdom. Highly deacetylated chitosan (e.g.,
> 85%) exhibits low degradation rate in aqueous media and
may last several months, and thus leads to great potential in the
development of inexpensive and versatile drug encapsulating
J. Med. Biol. Eng., Vol. 29. No. 6 2009 305
systems [12,13]. Its excellent gel-forming properties and ability
to be re-shaped into various forms simply by thermally induced
separation method strongly enhances its potential applications
in the biomedical field. Many researchers also found that
chitosan has osteoinduction and osteoconduction potentials
when used as a bone scaffold material [14,15]. As a
biodegradable material, chitosan had been given a lot of
attention in biomedical applications. Its blood clotting
characteristics made it well accepted as a wound healing agent,
skin grafting template and hemostatic agent [16-18]. In general,
it is also well-known to promote the formation of scaffolding
matrix in tissue regeneration process. On the other hand, among
many commercially available ceramics, CaSO4 is frequently
used as bone repairing and replacement material because of its
biocompatibility, bioactivity, mechanical strength, and
non-toxicity. From clinical experience, CaSO4 can be gradually
absorbed; that is followed by new bone formation yet without
compromising the required intimacy of bone-implant contact.
Kinetically, CaSO4 degraded faster than tricalcium phosphates
and have shown favorable osteoconduction and resorption
properties in animal studies. Also, CaSO4 can be resorbed and
replaced by new remodeled bone completely [19,20].
Consequently, we focused on the development of
bio-resorbable membrane prepared from chitosan and CaSO4,
and attempted to evaluate the feasibility of devising composite
membranes for GTR applications.
We previously reported that the chitosan membranes
prepared by thermally induced phase separation method
followed by treatment with non-toxic NaOH-gelating,
Na5P3O10- and Na2SO3- cross-linking agents exhibited similar
properties with chitosan membrane that had been strengthened
by glutaraldehyde as a cross-linking agent [12,15,21]. In this
study, we took advantage of the unique properties of chitosan
and CaSO4 to prepare a composite membranes system. To
retain the good biocompatibility of these two unique materials
within the same device, we used NaOH as the gelating agent in
the process of preparing CaSO4-chitosan composite membrane.
SEM observation was conducted to examine the morphology of
the membrane surface. Also, some fundamental properties of
the CaSO4-chitosan composite membranes were examined,
such as water content, mechanical strength and degradation. In
addition to assess the physical properties of CaSO4-chitosan
composite membranes, the biological function of the composite
membranes was studied by animal test. We wish to establish
the feasibility of composite membranes made of the two
naturally occurring biomaterials, CaSO4 and chitosan, for GTR
in periodontal application.
2. Materials and methods
Chitosan with molecular weight of 300,000 and
deacetylation degree of 83% was purchased from TCI
(Tokyo, Japan). Chitosan with molecular weight of 70,000
and deacetylation degree of 75% was purchased from Sigma
(St. Louis, MO). CaSO4 (Merck-Schuchardt, Germany) was
classified through a sieve with 0.104 mm openings. Acetic
acid was purchased from Sigma (St. Louis, MO). All
chemicals used in this study were of reagent grade.
2.1 Preparation of CaSO4-chitosan composite membranes
Chitosan was dissolved in acetic acid (0.1M) to prepare a
2% (w/v) chitosan solution. This chitosan solution was filtered
and mixed with CaSO4, weight of ratio CaSO4/chitosan 65:35,
and then stirred 24 hours. Each 19 ml of mixed solution was
poured into a 9-cm Petri-dish and placed in a drying oven at
40°C with proper ventilation for overnight. After drying, the
membranes were immersed in 0.1N NaOH 4 hours, and then
washed with distilled water and pressed from two sides with
polyethylene (PE) thin films, and then placed in oven (40°C)
until completely dry. The chitosan-containing CaSO4 membranes
exhibited ivory-like uniform and opaque texture. All prepared
chitosan membranes were stored in desiccators until use.
2.2 Water content measurement
The water content (W.C.) of the membrane was
determined by swelling the membrane in pH7.4 of phosphate
buffered saline (PBS) at room temperature. After the membrane
reached equilibrium state, the wet membrane was blotted with
filter paper to remove the water adhered on the surface. The
water content of the membrane was calculated as:
. . ( ) / 100%w d wW C W W W= − × (1)
where Ww and Wd are the weights of wet and dry membrane,
respectively. The experiment was conducted 3 times and a
mean and standard deviation were calculated.
2.3 Mechanical property measurement
The mechanical properties of the membranes were
measured in hydrated condition. The membranes, 1 cm × 6 cm,
were hydrated in 0.1 M pH 7.4 phosphate buffer before being
subjected to mechanical testing. The tensile strength
measurements of the membranes were charted up to the point
where they were broken. The mechanical parameters of these
chitosan membranes were calculated and recorded
automatically by using an MTS Systems (Eden Prairie, USA) at
a crosshead speed of 10 mm/min.
2.4 In vitro degradation test
The in vitro degradation tests of the prepared membranes
were conducted by incubating the membrane in 10 ml of pH
7.4 PBS on a shaker set at 40 rpm and 37°C. At predetermined
time intervals, the membrane was taken out of the incubation
medium, washed with distilled water, dried and its weight was
measured. Another fresh 10 ml PBS was added into the vial for
continuum degradation test. The degradation profiles were
expressed as the accumulated weight losses of the membrane.
2.5 SEM observation
The surface microstructure of membranes was examined by
scanning electron microscope (SEM). Before SEM observation,
all samples were dried, sputter-coated with gold, and examined
under a scanning electron microscope (JEOL, JSM-5300, Japan).
GTR with use of CaSO4-Chitosan Membrane 306
Figure 1. (a) Dental round burr. (b) Macroscopic appearance of an experimental site. A bone defect (6 mm in diameter) was created at skull of
a rabbit. A defect on the skull covered with (c) chitosan (70 kDa) membrane, (d) chitosan (30 kDa) membrane,
(e) CaSO4-Chitosan (70 kDa) composite membrane, and (f) CaSO4-Chitosan (300 kDa) composite membrane (10×10 mm2 in area).
Figure 2. SEM micrographs of the membrane: (a-b) a chitosan (70 kDa) membrane, (c-d) a chitosan (300 kDa) membrane membrane, (e-f)
CaSO4-Chitosan (70 kDa) composite membrane, and (g-h) CaSO4-Chitosan (300 kDa) composite membrane.
2.6 Animals and operation procedure
SD rats weighing about 200 g, fed with commercial
food and RO water, were included in this randomized,
blinded study. The rats were anesthetized by
trans-abdominal injection of Zoletil 50 (mixture of
Tiletamine and Zolezepam, 1:1) (0.2 ml/200 g). Following
the injection, the skull was shaved and the surfaces at
surrounding sides of the skull were exposed via
full-thickness incision. A man-made defect (6 mm in
diameter) was generated with a dental round burr (Figure
1(a)). Prior to implantation, the membranes were hydrated
in physiologic saline to restore their elasticity. The defect
was then covered with the membranes (Figures 1(b-e),
10 × 10 mm2 in area, 0.1 mm in thickness). In the control
group, the bone defect was not covered with any chitosan
membrane. The wounds were then carefully sutured. For
each animal with membrane and control, an initial healing
period of 4 weeks was allowed.
2.7 Histological preparation and evaluation
After the healing periods, the rats were sacrificed by
injecting an overdose of KCl into the heart. The skull tissue
containing bone defects was removed by a larger-size dental
trephine burr. The specimens of center of defect were fixed in
10% neutral-buffered formalin, decalcified in 10% formic acid
and then dehydrated in an ascending graded series of ethanol
solutions, and afterwards, embedded in paraffin. A series of
5-µm transverse sections encompassing the entire bone defect
specimen were prepared and stained with hematoxylin-eosin
and then subjected to light microscopic observation.
3. Results
3.1 Morphology of CaSO4-chitosan composite membrane
As indicated in Figure 2, the SEM micrographs showed
the characteristic morphological aspects of various chitosan
membranes. All chitosan membranes produced by thermally
J. Med. Biol. Eng., Vol. 29. No. 6 2009 307
(a)
(b)
Figure 3. EDX analysis of (a) CaSO4-Chitosan (70 kDa) and (b) CaSO4-Chitosan (300 kDa) composite membrane.
Table 1. Physical Properties of CaSO4/Chitosan CompositeMembranes (Average ± SD, n = 4).
Equilibrium water
content at 24 hr (%)
Young’s modulus
(MPa*)
Ultimate elongation
(mm)
Ultimate strength
(Pa)
CaSO4/70 kDa Chitosan 36.0 ± 2.4 24.7 8.0 ± 0.8 190.7 ± 11.7
CaSO4/300 kDa Chitosan 46.2 ± 0.7 18.8 21.6 ± 4.1 385.3 ± 63.0
Pure 300 kDa Chitosan 53.1 ± 1.1 15.0 10.9 ± 3.2 169.3 ± 64.7
*Chitosan membranes were hydrated in pH 7.4 PBS before experiment run.
induced phase separation demonstrated a dense
morphological structure. The roughness was visible on the
CaSO4-chitosan composite membranes. Contrarily, the pure
chitosan membrane showed a smoother surface as compared
with all CaSO4-chitosan composite membranes. Furthermore,
with elemental analysis using EDX (Figure 3), calcium could
be detected on the surfaces of the composite membranes.
Interestingly, the calcium ratio of the CaSO4-chitosan
(300 kDa) composite membrane surface was higher than that
of CaSO4-chitosan (70 kDa). It manifested that CaSO4 could
be “float” more on the surface of the membrane when using
300 kDa chitosan as the source of binding material.
3.2 Basic properties of CaSO4-chitosan composite membrane
When considering the development of guided tissue
regeneration barrier materials, the basic bulk and mechanical
properties ought to be reviewed. In addition, the appropriate
degradation rate of a material has to be evaluated to assess
whether it meets the requirements and diversities of tissue
regeneration procedures. Interestingly, CaSO4-chitosan
composite membranes have become very desirable elastomers
to biomedical engineers, as indicated by the wider range of
physical characteristics, especially with higher ultimate length.
As expected, the presence of CaSO4 would affect Young’s
modulus of the membranes in this study, as shown in Table 1.
The Young’s modulus of the membrane increased from 15.0
MPa (for pure chitosan) to 18.9 MPa CaSO4-chitosan
composite membrane.
Moreover, as shown in Figure 4, the CaSO4-chitosan
composite membranes showed a more rapid mass loss than the
pure chitosan membranes in 30-day shaking test. All
CaSO4-chitosan composite membranes degraded to about
30%–50% of initial weight after 30-day shaking test. For the
composite membranes, the degradation behavior showed a
typical biphasic profile. There was an initial rush of
degradation during the first 6 days of the study. This rapid
Mass
lo
ss (
%)
Time (days)
Figure 4. The degradation profile of the membranes in pH7.4 PBS
solution at 37°C for 30-day shaking.
degradation period probably represented the release of feebly
entrapped and surface-associated CaSO4. After the first 6
days, the composite membranes showed slower degradation
rates. However, the degradation rates were slightly different
between the composite membranes. The degradation of
CaSO4-chitosan composite membranes increased with the
decrease of the molecular weight of chitosan, probably due
to CaSO4 on the CaSO4-chitosan (300 kDa) composite
membrane surface being richer than that of CaSO4-chitosan
(70 kDa). As a result, in the first 6 days of degradation, the
calcium ion was released faster from the membrane surface.
Overall speaking, the CaSO4-chitosan (300 kDa) composite
membranes showed a more rapid mass loss. In other words,
the degradation result corresponded roughly to the EDX
analytical result.
Figure 5 shows the surface morphologies of the
CaSO4-chitosan composite membranes after 30 days of
sh ak in g . SEM examin a t ion s r evea l ed th at th ese
CaSO4-chitosan composite membranes still exhibited rough
GTR with use of CaSO4-Chitosan Membrane 308
Figure 5. SEM micrographs of the membrane: (a-b) a chitosan (70 kDa) membrane, (c-d) a chitosan (300 kDa) membrane membrane, (e-f)
CaSO4-Chitosan (70 kDa) composite membrane, and (g-h) CaSO4-Chitosan (300 kDa) composite membrane after 30 days of
shaking.
Mass
lo
ss (
%)
Time (days)
Figure 6. Water contact profile of the membranes in pH 7.4 PBS
solution in 10-minute period of experiment.
surfaces and had good integrity, albeit with signs of
degradation on the surface. CaSO4 was steadily released
and dissolved from the membranes while, concurrently,
chitosan was degraded only gradually. Relatively speaking,
the surface of neat chitosan retained an intact and smooth
surface morphology as before. As a guided regeneration
barrier material, the appropriate degradation rate of
material ought to be managed to fit into the schedule of
remodeling of tissue regeneration. Although, the resorption
process could be further facilitated by enzyme digestion in
real applications, we suggested that the membranes
prepared in this study could reasonably meet the
degradation requirement of bioresorbable membrane used
for GTR from these in vitro observations.
Another important characteristic of these chitosan
membranes is that they could reach steady hydration
equilibrium state within the 10-minute period of the
experiment, as seen in Figure 6 showing water content
profiles. This rapid swelling phenomenon would be
beneficial to the clinical manageability and surgical
procedures. The bulk properties of prepared
CaSO4-chitosan composite membrane are summarized in
Table 1. The results indicated that the CaSO4-chitosan
composite membranes prepared in this study fulfilled the
requirements of bioresorbable membrane employed as GTR
material. In clinical practice, when materials are used as
tissue regeneration barrier membranes, they are generally
required to maintain certain barrier functions for 4 to 6
weeks in order to secure the successful restoration of
periodontal tissues. The mechanical strength test results
indicated that these membranes had appropriate mechanical
strength for the requirement of GTR.
3.3 Histological observations
Varying degrees of bone healing were observed beneath
the CaSO4-chitosan composite membranes in comparison to
the control group. Figure 7 shows a typical transverse section
of experimental bone defect 4 weeks after surgery. In the
control group, the connective tissue grew into the bone
defect area and prevented the bony cells from growing back
to their natural form or space and thus destroyed the healthy
process of new bone growth (Figure 7(a)). Interestingly, in
the experimental groups (i.e., defect covered with
CaSO4-chitosan (300 kDa), and CaSO4-chitosan (70 kDa)
composite membranes), the connective tissue cells had only
limited proliferation on their original sites and prevented the
connective tissue cells from intruding into the space of the
bone defect (Figures 7(b)-(e)). Sequentially speaking, the
bone defect was allowed with the space and critical healing
time to be repaired by newly yet slowly formed bone that
proliferated in regions partially or fully protected and
separated by the CaSO4-chitosan composite membrane. This
process practically prevented any connective tissue from
invading into the bone defect. Furthermore, no obvious
inflammatory response was observed around the chitosan
membrane at this initial healing stage. However, the bone
healing seemed to be slowed down while the chitosan
membrane still occupied and saved the space exclusively for
the bone tissue to be healed in this isolated area (as compared
to the control).
J. Med. Biol. Eng., Vol. 29. No. 6 2009 309
(a)
(b)
(c)
(d)
(e)
Figure 7. Histological section of skull portion of a rat undergoing GTR procedure 4 weeks after surgery; (a) control, without chitosan
membrane coverage, (b) with chitosan (70 kDa) membrane coverage, (c) with chitosan (300 kDa) membrane coverage,(d)
CaSO4-Chitosan (70 kDa) composite membrane coverage, and (e) with CaSO4-Chitosan (300 kDa) composite membrane coverage.
Hematoxylin and eosin stain; original magnification × 100.
The preliminary results demonstrated that these prepared
CaSO4-chitosan composite membranes in this study could
successfully isolate the bone defect from ingress of connective
tissue cells and provide a space where bony tissue cells could
grow into later. There are other advantages, including the
readily gel-forming properties, and being easily available in
pure form, biodegradable and osteoinductive. Full utilization
of the characteristics of chitosan could allow it to become a
promising material in GTR applications.
4. Discussion
Many aspects of technical development of bioresorbable
membrane materials for GTR applications are focusing on
their rigidity and degradation rate, and with special emphasis
on the ease of clinical manageability. Lots of effort has been
spent on improving the biological function of barrier
membrane. For example, surfaces coated with alginate were
found to resist cell adhesion. In this study, we brought together
two attractive biomaterials, CaSO4 and chitosan, which are in
abundance and inexpensive, and possess excellent
biocompatibility and biodegradability, in applications for GTR.
Consequently, we have presented here a simple phase-induced
separation method to prepare a series of CaSO4-chitosan
barrier membranes. Briefly, CaSO4 was annexed to chitosan
matrix to prepare the composite membranes with high
mechanical strength. Following the process, CaSO4 also
altered morphological structure and degradation behavior of
the chitosan membranes. These changes probably are
attributed the ability of CaSO4 to bind with chitosan through
ionic bonding, and subsequently, to cross-link to the different
parts of chitosan polymer chains. To verify this postulation, we
tested the membranes in a medium of 0.1 N acetic acid. We
observed that CaSO4-chitosan membranes were not dissolved
completely even after 24-hour of shaking (not shown),
whereas a neat chitosan membrane would be readily dissolved.
Subsequently, we applied these CaSO4-chitosan
composite membranes to the animal GTR study models.
GTR with use of CaSO4-Chitosan Membrane 310
Based on the findings from the histological evaluation, all
three prepared chitosan membranes apparently exhibited
better membrane integrity in bone defect healing after 4
weeks and provided good cell separation ability (see Figures
7(b)-7(e)) compared with controls (Figure 7(a)). Among the
chitosan membranes tested, the defects covered with
CaSO4-chitosan composite membranes had higher percentage
of new bone formation. On the contrary, in the case of the
control group, the connective tissue might have grown
preemptively into the bone defect area, and that caused the
bony cells, with much slower growth rate, to not be able to
grow back into their originally designated space and form.
Another important factor for the success of GTR techniques
is that the barrier material ought to withstand a period long
enough for the bony tissue to reach sufficient healing stages.
In clinical practice, the tissue regeneration barrier
membranes are generally required to maintain their barrier
functions for 4 to 6 weeks, with ample mechanical strength
left, in order to secure the restoration of periodontal tissues.
As observed from Figure 5, it could be concluded that these
chitosan membranes were apparently suited for GTR in
biodegradable characteristics.
5. Conclusion
On the basis of the results observed, it could be
concluded that the CaSO4-chitosan composite membranes
prepared in this study appeared to be of great promise for
application in GTR in general. However, to assess and
explore the full potential of these materials in physiologically
more demanding periodontal applications for GTR, we shall
need to perform extensive and in-depth studies of animal
models that more closely resemble to the human anatomy,
such as in the porcine oral cavities.
Acknowledgements
This work was supported by grants from the National
Science Council, Taiwan (94-2213-E-214-038, 96-2815-C-214
-018-E).
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