CN1259344C - Application of o-substituted carboxy methyl chitin as antiadhesion material in surgical operation - Google Patents

Abstract

The present invention discloses the application of O-substituted carboxy methyl chitin as anti-adhesion material in a surgical operation. The O-substituted carboxy methyl chitin has the following unit structural formula: O-substituted carboxy methyl chitin (O-CMC). Preparation is simple and feasible. The O-substituted carboxy methyl chitin has biodegradability. The O-substituted carboxy methyl chitin is easily dissolved in neutral water solution (whose pH is equal to 7.0). The O-substituted carboxy methyl chitin can be used in a human body in a solution mode, the O-substituted carboxy methyl chitin is directly coated on the surface of a wound, and the use of the O-substituted carboxy methyl chitin for the organ and the tissue whose appearances are complicated is convenient. Compared with N-CMC and NO-CMC, O-CMC has better biocompatibility (i.e. toxicity is minimum). The O-substituted carboxy methyl chitin has the appropriate inhibitory capability for the growth of fibroblast and has the weak function for the growth of epidermal cells. Animal experiments prove that in all carboxy methyl chitin with a substituted structure, the anti-adhesion effect of O-CMC is the best after a surgical operation.

Description

O-substituted carboxymethyl chitosan is in the application of surgery operation antiblocking material

Technical field

The present invention relates to cm-chitosan prevents adhesion material in surgical operation application.The invention still further relates to the purification process of cm-chitosan.

Background technology

In the postoperative agglutinations such as ambition, abdominal cavity, vertebra, tendon repair, oral cavity,, stay sequela such as pain, intestinal obstruction to patient because the hyperplasia and the infiltration of fibrous tissue around the otch can stick together inevitably.Solve adhesion not only simply but also reliably way be to adopt so-called " physical barriers ", promptly inert material is placed the cutting part of tissue, the growth of fibrous tissue is taken blocking effect.For a long time, external domestic gelfoams, autologous fat, silicone rubber membrane, the hyaluronic acids etc. of adopting prevent the generation of adhesion as isolated material more, but they all influence result of use because of separately limitation.Gelfoam can make inoblast spread along it or hinder fat to survive and make cicatricial adhesion more extensive; Liquefaction and necrosis can take place and bring out the scar generation in autologous fat, phenomenons such as hemotoncus infects and skin sink can occur for the district simultaneously; Silicone rubber membrane really can reduce the generation of scar and adhesion, but it can not absorb by body, implants the back as in the foreign matter extended residual body, can cause mechanical stimulus to health; The hyaluronate sodium coated is easy, but price is high, and late period, preventing adhesiving effect was also limited.Degradation material is the new ideas that propose recently, and it has implanted buffer action after operation, energy degraded and absorbed behind wound healing, can the long-term existence body in, be post-operation adhesion preventing Development of Materials direction.

Chitin formal name used at school β-(1,4)-2-acetylaminohydroxyphenylarsonic acid 2-deoxy-D-glucose belongs to the natural polymer of linear polyose.Chitin extensively is present in the crust of marine animals such as shrimp crab at the nature aboundresources, and chitosan is the product of chitin after taking off acetyl.Because the chitosan source is abundant, preparation is simple, low price, it obtains extensive use in industrial aspect such as weaving, papermaking, food, makeup, sewage disposals.Since the nineties, chitin and chitosan are more and more paid attention in medical circle as a kind of new biomaterial, chitosan at slowly-releasing control release thing carrier, can absorb aspects such as sutures, artificial skin and have wide practical use.

Chitosan is a brand-new research direction as the post-operation adhesion preventing material, is abroad also only just appearing in the newspapers recently.Chitosan is nontoxic, nonirritant, and no immunizing antigen is the good embedded material of biocompatibility; Chitosan is subjected to the effect of enzyme in vivo, and degraded generates nontoxic small molecules, is absorbed by organism easily; The selectivity that also has chitosan resists unique biological activity of human fibroblastic growth, these all be degradable post-operation adhesion preventing material of new generation the character that must have.The deficiency of chitosan is to be insoluble in water, can only make film, for the organ and the tissue of complex contour, and fixing comparatively difficulty.Solvability is greatly improved the chitosan of modifying through the carboxymethyl shell in water, can spray to wound surface, uses more convenient.The Canadian Dalhousie Kennedy of university in 1996 etc. are once N, and O-completes methyl chitosan as the post-operation adhesion preventing material, and they use cm-chitosan and handle wound in the abdominal cavity of mouse model.Experimental result shows that the size of adhesion area, intensity and quantity have all obtained effective minimizing.Costain is report then, and the different periods use that cm-chitosan are antiseized to be connected with different effects in the surgical procedure, operation promptly close the abdominal cavity at last before use the most effective.

The domestic in recent years research report of carrying out adherence preventing material has the trend that increases, as the degradable polyamino acid film prevention of employings such as the Chen Yuguang rabbit postoperative canalis spinalis adhesion (nineteen ninety-five) of Zhongshan Medical Univ.; The Lv Decheng of Dalian Medical Univ etc. use chitosan film prevention chicken unguiflexor tendon adhesion (1996); The Sun Kang of Dalian Medical Univ etc. has compared the effect (1996) of 5 kinds of material preventions such as polymer fiber element, gelfoam rat postoperative epidural adhesion; Personnel selection such as the Mei Baoshan of First People's Hospital, Hefei tire amnion prevention chicken postoperative adhesion of tendon (1997); No.1 Military Medical Univ.'s gold the earth etc. prevents new zealand rabbit canalis spinalis postoperative scar adhesion (1997) with the polylactide film; The Hou Xiao of Shanghai Second Emdical University chief and Fan Tianyou etc. prevent rabbit postoperative epidural adhesion (1998) with hyaluronate sodium; Safe good grade the in Shandong Medical University palace adopted pachydermia (1998 years) in body in clinical.The big Long March Hou Chunlin of hospital of two armies etc. adopt chitosan prevention adhesion of tendon (1999).The Bi Ying of University Of Qingdao etc. adopt the adhesion (2002) of hyaluronate sodium film prevention peritonaeum and large intestine serous coat.They studies show that: these materials all have certain preventing adhesiving effect, but also exist various limitation.

Develop a kind of new material that prevents tissue adhesion, alleviate patient's misery, reduce the surgical operation sequela, improving the operation quality is the important topic that urgent need solves.

Summary of the invention

The objective of the invention is to develop a kind of new material that prevents tissue adhesion, alleviate patient's misery, reduce the surgical operation sequela, improve the operation quality.

The present invention also aims to provide a kind of purification process of above-mentioned materials.

The present invention is the application of O-substituted carboxymethyl chitosan in surgery operation antiblocking material, and described O-substituted carboxymethyl chitosan has following modular construction formula:

The present invention is to O-substituted carboxymethyl chitosan, N-substituted carboxymethyl chitosan, N, and O-substituted carboxymethyl chitosan carries out following simultaneous test respectively, and its each character is analyzed:

1, items such as alkali titration, colloidal titration, infrared spectra, nucleus magnetic resonance, ultimate analysis, viscosity are analyzed, and have determined the structural parameter such as molecular weight, deacetylation, degree of substitution by carboxymethyl and the position of substitution of cm-chitosan, see Fig. 1, shown in Figure 2, and table 1:

Table 1

Sample	Deacetylation	Viscosity-average molecular weight * 10 5	Total substitution value		O position substitution value		N position substitution value
									Potentiometric titration	Colloidal titration	Potentiometric titration	Colloidal titration	Potentiometric titration	Colloidal titration
			Chitosan	0.87	5.67
O-CMC	0.87	3.15							0.75	0.79	0.75	0.79	-	-
			N-CMC	0.87	5.89	0.56	0.41	-							-	0.56	0.41
N，O-CMC-1	0.87	5.12							0.56	0.38	0.19	0.1	0.37	0.28
			N，O-CMC-2	0.68	2.89	0.75	0.64	0.35							0.26	0.4	0.39
N，O-CMC-3	0.68	3.62							0.84	0.75	0.34	0.31	0.5	0.44
			N，O-CMC-4	0.79	3.96	0.93	0.86	0.61							0.32	0.61	0.54
N，O-CMC-5	0.87	3.31							1.18	1.09	0.46	0.33	0.72	0.76

Quality index is as follows:

Outward appearance: white or light yellow chip solid, soluble in water, aqueous solution neutrality

Molecular weight is 5～500,000,

The deacetylation of chitosan is about 70～80%,

The substitution value of cm-chitosan is greater than 65～80%,

Pb content is 0.00001～0.000065%,

As content＜0.00001%.

2, the analysis of material biological degradability:

The in vitro tests result of N,O-Diacetylmuramidase shows that all cm-chitosan all have biological degradability, and its biodegradation rate, biological degradability are influenced by chemical structure and molecular weight.

3, the looks dissolubility of material experiment:

With reference to ISO10993-1:1992 medicine equipment biological assessment standard and requirement, difference is replaced CMC carried out cell toxicity test, the test of acute general toxicity, intracutaneous irritant test, allergy and pyrogenic test.

A) cell toxicity test:

O-CMC and N-CMC do not constitute infringement to the L929 cellular form of vitro culture, and the growth of pair cell and propagation all do not have obvious restraining effect, and cell proliferation index percentage and blank group be there was no significant difference (P＞0.05) relatively.And N, O-CMC has cytotoxicity in various degree along with the difference of substitution value.

B) acute general toxicity test:

Movable feed of the mouse of the CMC experimental group of O-CMC, N-CMC, hyaluronic acid and degree of substitution by carboxymethyl higher (＞70%) and stool and urine are normal, and the mental status is good, toxic reactions such as no convulsions, paralysis, respiration inhibition.The physique amount all has increase in various degree.The CMC of substitution value lower (about 50%) and the mouse movement of chitosan experimental group reduce, and listless, body weight all has slight decline, belongs to mild to moderate poisoning.Do not get rid of the influence of residual Mono Chloro Acetic Acid and adsorbed impurity.Both acute general toxicities reduce after dialysing, and its acute general toxicity awaits further determining.

C) intracutaneous irritant test:

Rabbit test all samples side and control sides were kept the score and are 0 in 1,24,48 hours, did not all have erythema or oedema phenomenon, showed that the stimulus intensity that is tried thing is a nonirritant.

D) eye conjunctiva irritant test:

No matter all given the test agent are once or repeatedly eye irritant tests to the influence of rabbit eyes, and each test duration eye droppings irritant reaction of its conjunctiva, iris, cornea and contrast eye is kept the score and is 0, shows and is tried the thing multiple dosing to the eye nonirritant.

E) pyrogen testing:

Behind every rabbit injection O-CMC, fervescence all surpasses 0.6 ℃, and the summation that 3 rabbit body temperatures raise is lower than 1.4 ℃, and the O-CMC nonpyrogenic is described.

F) sensitivity test:

Undertaken by " pharmacological experiment ", the sensitivity test of experimental group injection liquid is through inducing injection and exciting 2 stages, in each group and the negative control group, cavy does not have anaphylaxis such as perpendicular hair, sneeze, retch, cough, expiratory dyspnea, hello sound, tic, collapse or death, tried material and under this test conditions cavy is not shown sensitivity response, reaction is kept the score and is 0 fen.Show that being tried material does not show sensitivity response to cavy under this test conditions; And positive controls (10% Protalbinic acid) is all in excitation phase death.

Above result shows, O-CMC and N-CMC no cytotoxicity.The CMC that substitution value is lower and the acute general toxicity of chitosan may be relevant with residual Mono Chloro Acetic Acid.The higher CMC of O-CMC, N-CMC and substitution value does not have acute general toxicity, to rabbit local skin and eye nonirritant, the healthy guinea pig whole body is not had anaphylaxis.The reaction of rabbit auricular vein injection O-CMC pyrogen-free.Confirm that O-CMC has excellent biological compatibility.

4, material is to the evaluation of body cell effect:

As Fig. 3, Fig. 4, Fig. 5, Fig. 6, shown in Figure 7, the result shows that cm-chitosan all has the different restraining effect of degree to inoblast and epidermal growth, and its restraining effect is subjected to the influence of chemical structure and material concentration.Table 2 is the influence to fibroblast proliferation of different concns CMC and hyaluronate sodium (HA).

Table 2

Drug level	Inhibiting rate (%)
								mg/mL	N-CMC	O-CMC	N，O-CMC-1	N，O-CMC-2	N，O-CMC-3	N，O-CMC-4	HA
8	25.3	41.1	73.9	97.3	89.1	19.9	17.5
								4	16.9	34.7	75.0	95.6	88.0	22.8	21.3

2	14.1	37.1	63.7	95.6	53.0	11.7	16.4
								1	6.1	40.2	43.7	10.4	10.4	2.9	6.0
0 (control group)	0	0	0	0	0	0	0

HA and various replacement CMC have restraining effect to fibroblasts proliferation, but CMC promptly can be suppressed to fibrocellular propagation in lower concentration (1mg/mL).HA and CMC are suppressed to the characteristic of fibroblast growth, can reduce the formation of the local collegen filament of wound, reach prevention of postoperative tissue adhesion, reduce the purpose of scar, and compare with HA, and CMC has the effect of stronger inhibition fibroblast proliferation.

Table 3 is different concns CMC and the HA effect to epidermal cell proliferation, the N of various substitution values, and O-CMC is the strongest to the epidermic cell inhibition of proliferation; N-CMC and HA to the epidermic cell inhibition of proliferation a little less than; O-CMC has only more weak inhibition to epidermal cell proliferation under the high density, and O-CMC also has a little promoter action under the lower concentration to epidermal cell proliferation.Can influence the healing rate of wound too by force to the effect of epidermic cell inhibition of proliferation, this respect O-CMC should be better than N, O-CMC.

Table 3

Drug level	Inhibiting rate (%)
								mg/mL	N-CMC	O-CMC	N，O-CMC-1	N，O-CMC-2	N，O-CMC-3	N，O-CMC-4	HA
8	38.9	35.0	77.8	69.0	86.4	75.2	41.5
								4	13.8	19.5	73.2	68.0	81.0	39.0	16.3
2	1.7	-11.3	65.8	64.8	67.4	4.1	17.3
								1	0.5	-5.9	32.3	51.2	57.3	6.2	10.7
0 (control group)	0	0	0	0	0	0	0

O-substituted carboxymethyl chitosan (O-CMC), the preparation simple possible, has biological degradability, be dissolved in neutral aqueous solution (pH=7.0) easily, can use in vivo in the solution mode, directly coated is to wound surface, and convenient especially for the use of the organ of complex contour and tissue, this is that it is than the more excellent part of chitosan.Compare with NO-CMC with N-CMC, O-CMC has better biocompatibility (being that toxicity is minimum), and growth of fibroblasts is had suitable inhibition, and the growth of epidermic cell is had weak effect.Experimentation on animals proves that O-CMC replaces in the cm-chitosan of structure at all, and its postoperative prevents that the adhesion effect from being best.

Description of drawings

Fig. 1 is chitosan and different substituted carboxymethyl chitosan infrared spectrograms.

Fig. 2 is an O-cm-chitosan nmr spectrum.

Fig. 3 is the light microscopic photos of embodiment 2 blank groups to the fibroblastic growth influence.

Fig. 4 is the light microscopic photos of embodiment 2 hyaluronic acid groups to the fibroblastic growth influence.

Fig. 5 is the light microscopic photo that the N-CMC group influences fibroblastic growth among the embodiment 2.

Fig. 6 is N among the embodiment 2, and the O-CMC group is to the light microscopic photo of fibroblastic growth influence.

Fig. 7 is the light microscopic photo that the O-CMC group influences fibroblastic growth among the embodiment 2.

Fig. 8 is the photo of 7 days posterior abdominal walls of O-CMC group among the embodiment 3.

The photo of 7 days posterior abdominal walls of Chitosan group among Fig. 9 embodiment 3.

The photo of 7 days posterior abdominal walls of Figure 10 embodiment 3 empty groups.

Embodiment

Embodiment 1

The synthetic preparation of cm-chitosan:

A) O-cm-chitosan (O-CMC)

Chitin 5 gram adds an amount of 50%NaOH solution and a small amount of Sodium dodecylbenzene sulfonate, alkalizes 1～2 day in-18 ℃, and elimination NaOH adds Mono Chloro Acetic Acid 22 grams, Virahol 80mL and small amount of N aBH ₄, stirring at room 3 hours, 60 ℃ of constant temperature 3 hours.Filter, precipitation is used 80% washing with alcohol, uses 95% alcohol flushing again, removes unreacted Mono Chloro Acetic Acid, gets the O-carboxymethyl chitin after the drying.In above-mentioned O-carboxymethyl chitin, add 60%NaOH solution 90mL, 80 ℃ were reacted 0.5 hour down, and 120 ℃ were reacted 1.5～2 hours again.The supernatant liquor that inclines adds suitable quantity of water in the viscous material, dissolving is filtered, and acetate transfers to neutrality, use 95% alcohol precipitation, filtration.Dehydrated alcohol and washing with acetone 2～3 times, drying gets white fiber shape solid O-CMC.

B) N-cm-chitosan (N-CMC)

1 gram chitosan, adding distil water 150mL, oxoethanoic acid 0.5 gram (chitosan: the mol ratio of oxoethanoic acid is 1: 1.2), room temperature reaction 1.5 hours, the pH value of this moment is about 3.6.Transfer to pH value 4～5 with 10%NaOH, gradation drips 10%NaBH ₁Solution 5mL reacted 2 hours again.Transfer to neutrality with dilute hydrochloric acid, 95% ethanol fully precipitates, and filters, and dehydrated alcohol and acetone rinsing get white powder N-CMC after the drying.

C) N, and the O-cm-chitosan (N, O-CMC)

Chitosan powder (deacetylation is 87.29%) adds certain density NaOH solution, and normal temperature alkalized 2 hours down.Gradation adds Mono Chloro Acetic Acid/aqueous isopropanol, and 70 ℃ were reacted 6～8 hours.Cooling, inclining supernatant liquor, adds suitable quantity of water in viscous material, stirring and dissolving, 10% hydrochloric acid transfers to neutrality.The elimination insolubles adds an amount of 95% ethanol sedimentation in the filtrate, filters, and the dehydrated alcohol flushing, oven dry gets white solid N, O-CMC.

Embodiment 2

The purifying of O-CMC is pressed step and is carried out:

1. add an amount of distilled water among the thick O-CMC, dissolving is filtered, and filtrate transfers to neutrality with acetate, uses 95% alcohol precipitation, filter, and dehydrated alcohol and washing with acetone 2～3 times, purpose is to remove unreacted alkali and Mono Chloro Acetic Acid, and drying obtains white fiber shape solid.

2. above-mentioned crude extract is added an amount of water and dissolve again and separate, in crude extract: the ratio of gac=1: 3 adds 30 purpose gacs, and purpose is to remove pyrogen and pigment.With the membrane filtration of 8 μ, remove gac and the unreacted chitin of small part after 4 hours.

3. be that the dialysis membrane of 10000MWCO is dialysed to distilled water with above-mentioned filtrate 2. with the molecular weight cutoff value, obtain white solid after the lyophilize.

4. under aseptic condition, above-mentioned steps O-cm-chitosan 3. is dissolved in again is made into 2% solution in the distilled water, with the membrane filtration of 0.45 μ, can implant the O-CMC solution of usefulness.

Embodiment 3

N-CMC, N are used in operation to rat ileum, and O-CMC-2, O-CMC, HA and blank group compare test.

Table 4 be document Belluco standard scoring (Belluco Cetal, J Surgica Reseach, 2001,100:217-221).

Table 4

The adhesion feature	Keep the score
		Adhesion area percentage 0＜25%＜50%＜75%＜100%	0 1 2 3 4
The adhesion kind
		No adhesion film, transparent, no blood vessel is opaque, or it is translucent, no blood vessel is opaque, exist capillary vessel opaque, have big blood vessel	0 1 2 3 4
The toughness of adhesion
		The easily separated adhesion of no adhesion adhesion itself can be drawn separation of synechia needs sharp weapon to separate	0 1 2 3

Animal model: the SD rat, middle lower abdomen is opened abdomen, cause apart from otch 1cm left side stomach wall damaged, area 1 * 2cm, the back proposes caecum, to the scratch of mesentery side intestines wall, area 1 * 2cm, use test materials 2mL in the wound, abrasive caecum face and abdominal-wall defect face are placed in opposite directions, close abdomen.Put to death animal after 7 days, the toughness of vessel growth degree and adhesion organization in the area of inspection adhesion, the adhesion organization.According to table 4 standards of grading, the high more expression adhesion of score is serious more.What adhesion was the most serious got maximum total points 11 minutes, if during no adhesion, minimum total points is 0 minute.

Table 5 is the CMC of various replacement structures and the preventing adhesiving effect of HA.

The preventing adhesiving effect evaluation of table 5CMC

Material	N-CMC	N，O-CMC-2	N，O-CMC-5	O-CMC	HA	Blank
							The scoring of animal number	n＝10 6.6±2.5	n＝10 5.9±2.3	n＝10 -	n＝10 4.9±1.6	n＝10 6.4±3.1	n＝10 10.0±1.2

P＜0.001, the difference significance

N, dead 4 of O-CMC-2 groups at duration of test, reason is that toxicity is excessive, no statistics, this group at first is eliminated.The result that keeps the score shows that O-CMC group total points is minimum, N, and the total points of O-CMC group, N-CMC group increases successively, and the HA group is close with the total points of N-CMC group, illustrates that the preventing adhesiving effect of O-CMC is best in the different materials.

Table 6 is O-CMC, Chitosan, physiological saline and barren preventing adhesiving effect:

The preventing adhesiving effect evaluation of table 6 O-CMC

Material	O-CMC	Chitosan	Physiological saline	Blank
					The scoring of animal number	n＝21 4.62±2.9	n＝17 5.76±2.3	n＝10 8.7±2.2	n＝30 9.6±1.8

P＜0.001, the difference significance

The result that keeps the score shows that O-CMC group score is minimum, and Chitosan group score is high slightly, and the physiological saline group is higher, and the score of blank group is the highest, and the preventing adhesiving effect that further specifies O-CMC is better than Chitosan.

Fig. 8, Fig. 9 and Figure 10 show the O-CMC group respectively, Chitosan group and blank group be in the adhesion situation of 7 days posterior abdominal walls of test, the adhesion of O-CMC group seldom, the adhesion of Chitosan group is more, the adhesion of blank group is very serious, and the antiblocking effect of visible O-CMC is best.

Claims (1)

1, O-substituted carboxymethyl chitosan is in the application of surgery operation antiblocking material, and described O-substituted carboxymethyl chitosan has following modular construction formula:

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