HYALURONAN – DOXYCYCLINE GELS PREPARATION

A

viscosity

B

HA mg/ml

0 2 4 6 8 10 12 14 16

0 5000 10000 15000 20000 25000 30000

] cP η[

HA mg/ml

0 2 4 6 8 10 12 14 16

6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4

Fig. 5 Effect of HA concentration on the influence of (A) viscosity and (B) pH.

The different concentration of powder HA ( 0.5, 1, 4, 10, 15 mg/ml ) were dissolved in pH 7.5, 50 mM phosphate buffer, the (A) viscosity and (B) pH were measured in 25 ℃.

Fig. 6 Effect of pH on the critical DC concentration to begin the precipitation at 0.1 mg/ml HA. The experiments were performed at pH 3.5 and 5.5. Temperature: (A) 2

℃

(B) 25

℃

(C) 37

℃

.

The powder HA was dissolved in pH 3.5 or pH 5.5 50 mM phosphate buffer, HA concentration was 0.1 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 3.5 or pH 5.5 50 mM phosphate buffer witout DC as blank, and measure the turbidity at 600 nm under controlled temperature (A) 2℃ (B) 25℃ (C) 37℃.

Fig. 7 Effect of pH on the critical DC concentration to begin the precipitation at 0.5 mg/ml HA. The experiments were performed at pH 3.5 and

5.5. Temperature: (A) 2

℃

(B) 2 5

℃

(C) 37

℃

.

The powder HA was dissolved in pH 3.5 or pH 5.5 50 mM phosphate buffer, HA concentration was 0.5 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 3.5 or pH 5.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (A) 2℃ (B) 25℃ (C) 37℃.

Fig. 8 Effect of pH on the critical DC concentration to begin the precipitation at 1 mg/ml HA. The experiments were performed at pH 3.5 and 5.5.

Temperature: (A) 2

℃

(B) 25

℃

(C) 37

℃

.

The powder HA was dissolved in pH 3.5 or pH 5.5 50 mM phosphate buffer, HA concentration was 1 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 3.5 or pH 5.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (A) 2 (B) 25 (C) 37 .℃ ℃ ℃

Fig. 9 Effect of temperature on the critical DC concentration to begin the precipitation at pH 3.5. The experiments were performed at 2, 25, 37 ℃. HA

concentration: (A) 0.1 (B) 0.5 (C) 1 (D) 5 mg/ml.

The powder HA was dissolved in pH 3.5 50 mM phosphate buffer, HA concentration was 0.1, 0.5, 1, 5 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 3.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (2, 25 or 37 ).℃ The concentration of HA in each figure was (A) 0.1 mg/ml (B) 0.5 mg/ml (C) 1 mg/ml (D) 5 mg/ml.

Fig. 10 Effect of temperature on the critical DC concentration to begin the precipitation at pH 5.5. The experiments were performed at 2, 25, 37 ℃. HA

concentration: (A) 0.1 (B) 0.5 (C) 1 mg/ml.

The powder HA was dissolved in pH 5.5 50 mM phosphate buffer, HA concentration was 0.1, 0.5, 1 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 5.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (2, 25 or 37 ).℃ The concentration of HA in each figure was (A) 0.1 mg/ml (B) 0.5 mg/ml (C) 1 mg/ml

Fig. 11 Effect of HA concentration on the critical DC concentration to begin the precipitation at pH 3.5. The experiments were performed at 0.1, 0.5, 1, 5 mg/ml HA. Temperature: (A) 2 ℃ (B) 25 ℃ (C) 37℃.

The powder HA was dissolved in pH 3.5 50 mM phosphate buffer, HA concentration was 0.1, 0.5, 1, 5 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 3.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (2, 25 or 37 ).℃ The turbidity were measured in temperature (A) 2℃ (B) 25℃ (C) 37℃ separately.

Fig. 12 Effect of HA concentration on the critical DC concentration to begin the precipitation at pH 5.5. The experiments were performed at 0.1, 0.5, 1 mg/ml HA. Temperature: (A) 2 ℃ (B) 25 ℃ (C) 37℃.

The powder HA was dissolved in pH 5.5 50 mM phosphate buffer, HA concentration was 0.1, 0.5, 1 mg/ml, then added different consistency of DC (0- 30 mM). We used the pH 5.5 50 mM phosphate buffer without DC as blank, and measure the turbidity at 600 nm under controlled temperature (2, 25 or 37 ).℃ The turbidity were measured in temperature (A) 2℃ (B) 25℃ (C) 37℃ separately.

Fig. 13 Effect of HA concentration on the critical DC concentration to begin the change of pH. The experiments were performed at (A) pH 3.5, (B) pH 5.5 phosphate buffer.

The different concentration of powder HA ( 0.1, 0.5, 1 or 5 mg/ml ) were dissolved in pH 3.5 or pH 5.5 50 mM phosphate buffer with different concentration of DC (0- 30 mM ). We used the pH 3.5 or pH 5.5 50 mM phosphate buffer without DC as blank. The turbidity were measured in 25 ℃; the phosphate buffer solution were (A) pH 3.5 and (B) pH 5.5.

Fig. 14 Effect of temperature on the critical DC concentration to begin the precipitation and viscosity. The experiments were performed at pH 3.5. (A)

turbidity (B) viscosity.

Reagents were dissolved in pH 3.5, 50 mM phosphate buffer with different concentration of DC at 10 mg/ml HA. Here we used CR model (control rate model) to detect the variation of viscosity. Data showed the detectable numeric of viscosity under smallest shear rate. Because the viscosity was too higher to measure the turbidity at 2 ℃, it was performed when temperature was controlled to be 25 and 37

℃. The control turbidity of HA was 10 mg/ml without DC. Viscosity was measured at 25 ℃. Here showed (A) the turbidity (B) the viscosity of the HA-DC structure.

A

viscosity

B

Zinc/DC mixing mole ratio

0 20 40 60 80 100 120

Zinc/DC mixing mole ratio

0 20 40 60 80 100 120

Fig. 15 Effect of zinc/DC mole ratio on the influence of viscosity and pH at 6 and 8 mg/ml HA. (A) viscosity (B) pH.

The reagent was dissolved in pH 7, 50 mM phosphate buffer. Different concentration of zinc/ DC mole ratio was dissolved in 6 or 8 mg/ml HA. Here we used CR model (control rate model) to detect the variation of viscosity. Data showed the detectable numeric of viscosity under smallest shear rate. The temperature was controlled to be 25 ℃. Original pH of HA was 7.2, viscosity was 110 and 158. Here it showed (A) the viscosity (B) the pH of the HA-DC gels.

Fig. 16 Effect of zinc/DC mole ratio on the influence of viscosity and pH at 4 mg/ml HA. (A) viscosity (B) pH.

The reagent was dissolved in pH 7, 50 mM phosphate buffer. Different concentration of zinc/ DC mole ratio (0, 05, 0.1, 0.5, 1, 5) was dissolved in 4 mg/ml HA. Here we used CR model (control rate model) to detect the variation of viscosity. Data showed the detectable numeric of viscosity under smallest shear rate. The temperature was controlled to be 25 ℃. Original pH of HA was 6.25, viscosity was 240. Here it showed (A) the viscosity (B) the pH of the HA-DC gels.

Fig. 17 Effect of different concentration of DC influenced the viscosity, turbidity and pH at pH 7.0. (A) viscosity, (B) turbidity and (C) pH.

Different concentration of DC (0, 25, 50, 100 μg/ml) was dissolved in Artz-Dispo with zinc chloride was under 0.5 Zinc/DC mole ratio; here we used CR model (control rate model) to detect the variation of viscosity. Data showed the detectable numeric of viscosity under smallest shear rate. The temperature was controlled to be 25 ℃. Original pH of Artz-Dispo was 7.25, viscosity was about 1650. Here it showed (A) the viscosity (B) the turbidity (C) the pH of the HA-DC gels. Each group was compared with control group. (* = p<0.05, * * = p<0.01).

Table 6-1 Comparative elastoviscosity of HA-DC gel and HA-DC structure with different concentration of DC.

With different concentration of DC, we used the HAAKE rotation rheomoter RS-1 to measure their elastoviscosity characteristic of each group. The pH and temperature (pH≒ 7, 37 ℃) conditions was under control for imitating the basic state of warm-blooded animal.

There were two groups HA-DC gel and HA-DC structure. The unequivocal different of them both was existing divalent metal (zinc chloride) or not.

The average molecular weight of the Artz-Dispo was 0.6-1.2 million, and the doxycycline concentration were 0, 25, 50 100 μg/ml. The dynamic shear modulus, G’

(elasticity- storage modulus) and G” (viscosity- loss modulus), were determined using the oscillation model. Measurements were made at a frequency range of 0.01 to 10 Hz. Here showed the average value of tree time measurement; each experiment was practiced under the same condition.

G’ at Hz (Pa) G’’ at Hz (Pa)

0.01 0.1 0.5 2.5 0.01 0.1 0.5 2.5

HA 0.020 0.056 0.435 7.056 0.071 0.763 4.160 13.170 Gel 25 0.055 0.238 1.761 8.473 0.093 0.801 4.749 13.716 Gel 50 0.052 0.347 2.302 8.574 0.261 0.839 4.910 13.970 Gel 100 0.338 1.166 2.796 10.363 1.087 2.055 6.492 16.895 DC 25 0.001 0.053 1.941 3.721 0.045 0.602 3.846 9.636 DC 50 0.004 0.187 1.263 3.969 0.049 0.489 3.160 10.004 DC 100 0.003 0.012 1.202 2.750 08.041 0.487 3.691 10.474

6.2 In vitro cell experiments

A

0 20 40 60 80 100 120 140 160

0 0 0.1 1 10 25 50

DC μg/ml

Cell viabillity (% of control)

IL-1 β 2ng/ml + + + + + +

B

0 20 40 60 80 100 120

0 1 10 25 50

DC μg/ml

Cell viabillity (% of control)

Fig. 18 Effect of DC on the influence of cell survival in human chondrosarcoma cells SW1353 (A) with or (B) without IL-1 β.

(A) The cells were pretreated with various concentrations of DC (0.1, 1, 10, 25, 50 μg/ml) for 8 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group. Three independent experiments were performed and those survival cells were detected by WST-1.

Each column and vertical line indicates the means and ± S.E. of each test. The relative amounts of survival cells were quantified by taking the control group as 100 %. (* = p<0.05,

* * = p<0.01)

(B) The cells were incubated with various concentration of DC (0, 1, 10, 25, 50 μg/ml) for 24 hours. Three independent experiments were performed and the survival cells were detected by WST-1. Each column and vertical line indicates the means and ± S.E. of each test; the relative amounts of survival cells were quantified by taking the control group as 100 %. (* = p<0.05, * * = p<0.01).

0 20 40 60 80 100 120 140

0 0 0.1 1 10 25 50

DC μg/ml

% of positive control

IL-8

IL-β ng/ml + + + + + +

Fig. 19 Effect of DC on the influence of the gene expression of IL-8, MMP-1, MMP-3 and MMP-13 in human chondrosarcoma cells SW1353.

The cells were pretreated with various concentration of DC (0.1, 1, 10, 25, 50 μg/ml ) for 8 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group to induce exhibition of cell inflammatory for observing the final gene expression of IL-8, MMP-1, MMP-3 and MMP-13 via RT-PCR as described under “Material and Methods.” The results showed the representative of three independent experiments, and the mRNA copy numbers were normalized against the corresponding copy number of GAPDH mRNA. The gene expression of each group was quantitated, and the relative expressions of mRNA were quantified by taking the IL-1 β -treated group as 100 %. Each column and vertical line indicates the means and ± S.E. of each group,. (* = p<0.05, * * = p<0.01, compare with the IL-1 β -treated group).

0 20 40 60 80 100 120 140

0 0 0.1 1 10 25 50

DC μg/ml

% of positive control

TYPE-II

IL-β ng/ml + + + + + +

* *

Fig. 20 Effect of DC on the influence of the gene expression of type II collagen and in human chondrosarcoma cells SW1353.

The cells were pretreated with various concentration of DC (0.1, 1, 10, 25, 50 μg/ml ) for 8 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group to induce exhibition of cell inflammatory for observing the final gene expression of type II collagen via RT-PCR as described under “Material and Methods.” The results showed the representative of three independent experiments, and the mRNA copy numbers were normalized against the corresponding copy number of GAPDH mRNA. The gene expression of each group was quantitated, and the relative expressions of mRNA were quantified by taking the IL-1 β -treated group as 100 %. Each column and vertical line indicates the means and ± S.E. of each group,. (* = p<0.05, * * = p<0.01, compare with the IL-1 β -treated group).

A

(a) 0 mg/ml (b) 0.01 mg/ml

(e) 4 mg/ml

B

0 20 40 60 80 100 120 140

0 0.01 0.1 1 4

Artz-Dispo mg/ml

Cell viabillity (% of control)

* *

C

50 60 70 80 90 100 110 120 130

0 0 10 50 100 250

Artz-Dispo ug/ml

Cell viabillity (% of control)

IL-1B 2ng/ml + + + + +

Fig. 21 Effect of HA (Artz-Dispo) on the influence of cell morphology and proliferation in human chondrosarcoma cells SW1353. (A) cell morphology, (B) cell proliferation with or (C) without IL-1 β.

(A) The cells were incubated with various concentration of HA (Artz-Dispo) (0, 0.01, 0.1, 1, 4 mg/ml) for 24 hours, the represented photographs of each group were showed, and the cell morphology was photographed. Magnification 40X.

(B) The cells were incubated with various concentration of HA (Artz-Dispo) (0, 0.01, 0.1, 1, 4 mg/ml) for 24 hours. Three independent experiments were performed and those survival cells were detected by MTT. Each column and vertical line indicates the mean and

± S.E. of each test; the relative amounts of survival cell were quantified by taking the control group as 100. (* = p<0.05, * * = p<0.01, compare with IL-1 β -treated group except control group).

(C) The cells were pretreated with various concentrations of HA (Artz-Dispo) (0, 0.01, 0.1, 1, 4 mg/ml) for 24 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group. Three independent experiments were performed and those survival cells were detected by WST-1. Each column and vertical line indicates the means and ± S.E. of each test; the relative amounts of survival cells were quantified by taking the control group as 100 %. (* = p<0.05, * * = p<0.01)

A

(a) 0 mg/ml (b) 0.01 mg/ml

(e) 4 mg/ml

B

0 20 40 60 80 100 120 140 160 180 200

0 0.01 0.1 1 4

Artz - Dispo mg/ml

Cell viabillity (% of control)

** **

Fig. 22 Effect of HA (Artz-Dispo) on the influence of cell morphology and proliferation in primary human chondrocyte. (A) cell morphology, (B) cell proliferation.

(A) The primary cells were incubated with various concentration of HA (Artz-Dispo) (0, 0.01, 0.1, 1, 4 mg/ml) for 24 hours; the represented photographs of each group were showed, and the cell morphology was photographed. Magnification 40X.

(B) The primary cells were incubated with various concentration of HA (Artz-Dispo) (0, 0.01, 0.1, 1, 4 mg/ml) for 24 hours. Three independent experiments were performed and those survival cells were detected by MTT. Each column and vertical line indicates the mean and ± S.E. of each test, and the relative amounts of survival cell were quantified by taking the control group as 100. (* = p<0.05, * * = p<0.01, compare with IL-1 β -treated group except control group).

A

MMP-1 MMP-3 MMP-13

IL-8

GAPDH IL-1β ng/ml

HA μg/ml

10 50 100 250

2 2 2 2 2

0 20 40 60 80 100 120

0 0 10 50 100 250

HA μg/ml

% of positive control

IL-8

IL-β ng/ml + + + + +

* *

Fig. 23 Effect of HA on the influence of the gene expression of IL-8, MMP-1, MMP-3 and MMP-13 in human chondrosarcoma cells SW1353.

The cells were pretreated with various concentration of HA (0, 10, 50, 100, 250 μg/ml ) for 24 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group to induce exhibition of cell inflammatory for observing the final gene expression of IL-8, MMP-1, MMP-3 and MMP-13 via RT-PCR as described under “Material and Methods.” The results showed the representative of three independent experiments, and the mRNA copy numbers were normalized against the corresponding copy number of GAPDH mRNA. The gene expression of each group was quantitated, and the relative expression of mRNA were quantified by taking the IL-1 β -treated group as 100 %. Each column and vertical line indicates the means and ± S.E. of each group,. (* = p<0.05, * * = p<0.01, compare with the IL-1 β -treated group).

0 50 100 150 200 250

0 0 10 50 100 250

HA μg/ml

% of positive control

TYPE-II

IL-β ng/ml + + + + +

** *

* *

Fig. 24 Eeffect of HA (Artz-Dispo) on the influence of gene expression of type II collagen in human chondrosarcoma cells SW1353.

The cells were pretreated with various concentration of HA (0, 10, 50, 100, 250 μg/ml ) for 24 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group to induce exhibition of cell inflammatory for observing the final gene expression of type II collagen via RT-PCR as described under “Material and Methods.” The results showed the representative of three independent experiments, and the mRNA copy numbers were normalized against the corresponding copy number of GAPDH mRNA. The gene expression of each group was quantitated, and the relative expression of mRNA were quantified by taking the IL-1 β -treated group as 100 %. Each column and vertical line indicates the means and ± S.E. of each group,. (* = p<0.05, * * = p<0.01, compare with the IL-1 β -treated group).

0 20 40 60 80 100 120

0 7.1 14.3 28.6

DC μg/ml in Artz-Dispo

Cell viabillity (% of control)

Fig. 25 Effect of HA-DC gels on the influence of cell survival in human chondrosarcoma cells SW1353.

The cells were incubated with hyaluronan–doxycycline gels for 24 hours, and the final concentration of HA was 2.86 mg/ml included various concentration of DC (0, 7.1, 14.3, 28.6 μg/ml). Each well was filled with 500 λ medium at first, then added 200 λ gels (HA 10 mg/ml) which included different concentration of DC (DC 0, 25, 50, 100 μg/ml) to imitate the condition of our animal experiments later, to examine the cell survival influenced by gel.

Four independent experiments were performed and those survival cells were detected by MTT. Each column and vertical line indicates the mean and ± S.E. of each test, and the relative amounts of survival cell were quantified by taking the control group as 100. (* = p<0.05, * * = p<0.01, compare with IL-1 β -treated group except control group).

IL-1β ng/ml

DC μg/ml

1 10 25 50

2 2 2 2 2

GAPDH IL-8

MMP-13 MMP-3 MMP-1

0 20 40 60 80 100 120

0 0 1 10 25 50

DC μg/ml

% of positive control

IL-8

IL-β ng/ml + + + + +

Fig. 26 Effect of HA-DC gel on the influence of gene expression of IL-8, MMP-1, MMP-3 and MMP-13 in human chondrosarcoma cells SW1353.

The cells were incubated with hyaluronan–doxycycline gels; the concentration of HA was 250 μg/ml included various concentration of DC (0, 1, 10, 25, 50 μg/ml) for 24 hours, then added IL-1 β (2 ng/ml) for 6 hours except the control group to induce exhibition of cell inflammatory for observing the final gene expression of IL-8, MMP-1, MMP-3 and MMP-13 via RT-PCR as described under “Material and Methods.” The results showed the representative of three independent experiments, and the mRNA copy numbers were normalized against the corresponding copy number of GAPDH mRNA, the gene expression of each group was quantitated, and the relative expression of mRNA were quantified by taking the IL-1 β -treated group as 100 %. Each column and vertical line indicates the means and ± S.E. of each group,. (* = p<0.05, * * = p<0.01, compare with the IL-1 β -treated group).

6.3 In vivo animal experiments

0 0.5 1 1.5 2 2.5 3

0 3 6 9 12 14 day

DOX HA GEL NS

Fig. 27 Observation of general status and body weight measurement.

The animal general status of each group: (a) DC (doxycycline), (b) HA (Artz-Dispo), (c) Gel (hyaluronan–doxycycline gels) and (d) normal saline (placebo group), were observed once a day, and measured each rabbit body weight before the pain assessment.

The body weight were measured at day 0 for the first time ( before the OA-induced surgery), and day 3, 6, 9, 12, 14 (the last day). (a= p＜0.05 between control group and other group, b= p＜0.05 between DC group and HA, gel groups, c= p＜0.05 between HA group and gel groups). N = 8.

% weight distribution of left hind paw

DOX

Fig. 28 Effect of DC, HA, gel and NS on the influence of percentage distribution on left hind paw.

After intra-articularly injecting of different drugs, (a) doxycycline, (b) HA (Artz-Dispo), (c) hyaluronan–doxycycline gels and (d) normal saline (control group), the hind paw weight distribution of each hind paw was measured independently. Measurement was performed three times and the % weight distribution of left hind paw was calculated by the formula of “General Protocol of Animal Experiment - in vivo Experiment”. The pain assessment was performed at day 0 for the first time (before surgery), and day 1 (after the OA-induced surgery), 4, 7, 10, 13. We measured the pain assessment with each group at the same time. Each column and vertical line indicates the mean and ± S.E. of each test, every group were compared with each other at the same day. (a= p＜0.05 between control group and other group, b= p＜0.05 between DC group and HA, gel groups, c= p＜0.05 between HA group and gel groups). N = 8.

A.

DC group

B.

HA group

C.

gel group

D.

control group

Fig. 29 Macroscopical appearance of femur.

The macroscopical appearance of articular surfaces on the femoral condyle of the 4 groups (A) DC group, (B) HA group, (C) gels group, (D) control group (OA). The pictures were representative of each group.

A.

DC group

B.

HA group

C.

gel group

D.

control group

Fig. 30 Macroscopical appearance of tibia.

The macroscopical appearance of articular surfaces on the tibia plateau of the 4 groups- (A) DC group, (B) HA group, (C) gels group, (D) control group (OA). The pictures were representative of each group.

Table 6-2 Macroscopical examination: changes in mean tibial and femoral lesion scores.

Histopathological assessment was conducted according to the Table 2-2 [56]. The evaluation of the natural history of the induced osteoarthritis was based on five macroscopical parameters scored changes in mean tibial and femoral lesion scores. Each group were performed OA-induced operation with partial lateral meniscectomy and section of the fibular ligament in rabbits, and then four groups were treated with (A) DC, (B) HA, (C) gel (D) normal saline separately. Each form indicates the mean and range of every test.

All the samples were evaluated and checked by the same Dr. randomly and double blind.

Mean lesion scores (range) Osteophytes/ chondrocytes 1.17

(1-2) 0.86

(0-1) 0.71

(0-1) 1.43 (1-3) Fibrillation (surface fragmentation) 2.33

(2-3) Osteophytes/ chondrocytes 1.17

(1-2) Fibrillation (surface fragmentation) 1.83

(1-2) 1.43

Loss of superficial layer Erosion of cartilage Fibrillation Osteophytes Fissures

DOX

Loss of superficial layer Erosion of cartilage Fibrillation Osteophytes Fissures

DOX

Fig. 31 Macroscopic examination in (A) femur (B) tibia.

Histopathological assessment was conducted according to the Table 2-2 [56].

Specimen were scrutinized and gave suitable score in (A) femur and (B) tibia samples by using the special magnifying glasses used in surgery to survey the articular cartilage surfaces of the femoral of the 4 groups- (a) DC group, (b) HA group, (c) gels group, (d) normal saline (control group). Those evaluations of five items include different lesion degree were based on three macroscopic parameters: (1) the location, (2) the type, and (3) the size of the OA changes. All the samples were checked by the same Dr. randomly and double blind. Each column and vertical line indicates the mean and ± S.E. of each examination, every group was compared with each other at the same inspect item. (a= p＜

0.05 between control group and other group, b= p＜0.05 between DC group and HA, gel groups, c= p＜0.05 between HA group and gel groups). N = 8.

(a) DC (b) HA