WO2014186854A1

WO2014186854A1 - Process for obtaining stem-cell-enriched suture thread, threads obtained, suture kit and the uses thereof

Info

Publication number: WO2014186854A1
Application number: PCT/BR2014/000153
Authority: WO
Inventors: Joaquim Murray Bustorf SILVA; Bruno Bosch VOLPE; Ângela Cristina Malheiros LUZO
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2013-05-24
Filing date: 2014-05-12
Publication date: 2014-11-27

Abstract

The present invention relates to a process for obtaining stem-cell-enriched suture thread, the suture thread obtained, the suture kit and the uses thereof. The threads obtained and the suture kit assist in treatment, recovery and cicatrization and may be used in surgical procedures such as, for example, in plastic surgery and gastrointestinal fistulas, burns, fistulas in patients with Crohn's disease, and the like. The suture thread and kit may be used in various types of fistula and furthermore may be useful in cicatrization in other types of afflictions, such as burns, wounds in diabetic patients, in plastic surgery, inter alia.

Description

Field of obtaining enriched yarn with stem cells, yarns, yarn and their uses Field of the invention

The present invention is a process for obtaining stem cell enriched suture, obtained suture, suture kit and its uses.

The present invention has application in treatment, recovery and healing and may be employed in surgical procedures, such as plastic surgery and gastrointestinal fistulas, burns, fistulas in Crohn's disease patients, among others.

Fundamentals of the invention

Gastrointestinal fistulas are an abnormal communication between two epithelialized surfaces, through which drainage of digestive secretion (debit) occurs. Its onset represents a series of complications for the patient, and there are few surgical problems that need more attention than gastrointestinal fistulas. Its onset may occur due to poor surgical technique or as an evolution of inflammatory bowel diseases, especially in patients with Crohn's disease. Gastrointestinal fistulas cause a series of disorders to the patient, such as hydroelectrolytic disorders, infections, sepsis, skin lesions, malnutrition, digestive hemorrhage, intestinal obstruction, among others. The severity of all these disorders depends on a number of issues such as etiology, type of fistula, anatomical location, fistula output (amount and quality of fluid lost) and age of the patient.

The conventional treatments that exist today (nutritional control of the patient, drug treatment and surgery) to aid recovery and healing of patients with enterocutaneous fistulas do not have good patient recovery rates, and in many patients the fistula takes from 3 to 8 weeks to recover. its healing and there are still chances of this fistula opening again. Damian García-Olmo, et al. (2005) tested the treatment of fistulas in Crohn's disease patients with adipose-derived mesenchymal stem cell transplantation. The group injected 3 to 30 x 106 cells (depending on the time of culture used) around the fistula site. Nine patients with different types of fistulas were treated. 66% of the fistulas were considered cured at 8 weeks of treatment, 22% did not close and remained open, and 11% could not be evaluated because they were considered inadequate for treatment.

Regarding cell adhesion in sutures, Jacques P. Guyette, et al. (2012) adhered to human mesenchymal stem cells in biological sutures. The sutures used were 4 cm and were placed into a Silastic® tube to which 1 x 10 6 cells in suspension were added. Cell growth and adhesion were observed after 24 hours. Hoechst and Phalloidin stained immunofluorescence confocal microscopy images were performed to confirm cell adhesion. The results showed that an average 1,806 mesenchymal stem cells were adhered to each 2cm suture. The threads enriched with mesenchymal stem cells were tested in rat hearts. The group compared the efficiency of the enriched yarn together with a cell injection group directly at the site. As a result, the animal group where the cell wire was used had greater cell transfer to the lesion site.

Comparing the study of the above group with the object of the present invention the microscopy images show that there is a much larger number of cells adhered when using fibrin glue. The thread used by them was only 4 cm, being efficient in some situations but not for large sutures, because it is small in size. The suture thread of the present invention is 30 cm and is considered a large size suture thread. This way it can be used in various areas that want to heal and recover the site faster and more efficiently. Given the above it would be useful if the technique had a suture enriched with mesenchymal cells capable of assisting in the treatment, recovery and healing of patients with fistula, transfer mesenchymal stem cells to the site of injury, causing recovery and healing. advanced because these cells release immunomodulation factors. The thread can be used in various types of fistulas and can still be useful in healing other types of diseases, such as burns, diabetic wounds, plastic surgery, among others.

Brief Description of the Invention

The obtained threads and the suture kit help in the treatment, recovery and healing and can be used in surgical procedures, such as plastic surgery and gastrointestinal fistulas, burns, fistulas in Crohn's disease patients, among others.

Brief Description of the Figures

Figure 1 represents the statistical analysis comparing the three groups of the in vivo test (control, injection and wires) in relation to the evolution of the fistula area on the day of operation until the 21st day.

Figure 2 represents the statistical analysis comparing the evolution of the fistula area in the three in vivo test groups (control, injection and wires) on days 0, 12 and 21.

Figure 3 is an immunofluorescence confocal microscopy image. A: Control suture, without staining, showing the absence of cells. B: Control of marked cells (green). C: Arrow points to the suture wrapped by a large number of cells, 10x magnification D: Arrow points to the suture wrapped by a large number of cells, 20x magnification Figure 4 represents a scanning electron microscopy image. A: The yellow arrows show some cells above and between the suture filaments. B: The arrows and the red bow show the action of fibrin glue forming a cell layer along the wire.

Figure 5 is an image of a fistula control group animal. A: First day of fistula formation and well-infected region. B: 21st day, region is almost healed and with localized infection.

Figure 6 is an image of a fistula control group animal. A: First day of fistula formation and well-infected region. B: 21st day, fistula healing did not occur and region is well infected with high stool output.

Figure 7 is an image of a fistula control group animal. A: First day of fistula formation and no site infection. B: Day 21, the healing of the fistula was almost complete and the presentation of a small infection site.

Figure 8 is an image of an animal from the group presenting with fistula and mesenchymal cell application. A: First day of fistula formation and small site infection with low stool output. B: 21st day, the healing of the fistula was practically complete and the presentation of a small infection site.

Figure 9 is an image of an animal of the group using the present invention. A: First day of fistula formation and small site infection with low stool output. B: 21st day, the healing of the fistula was practically complete and the presentation of a small infection site.

Figure 10 is an image of an animal of the group using the present invention. A: First day of fistula formation and small site infection with low stool output. B: Day 21, the healing of the fistula was complete and the circle shows the area where the fistula was.

Detailed Description of the Invention The present invention is a process for obtaining stem cell enriched suture, obtained suture, suture kit and its uses.

The process for obtaining enriched suture comprises the following steps:

(a) thrombin and calcium chloride drip along the entire length of the suture;

(b) stem cell dripping;

c) Fibrinogen drip;

d) Addition of culture medium sufficient to cover the suture;

e) Addition of 10% serum to the amount of culture medium added, and

f) Keep for a period between 2 and 24 hours in a CO ₂ greenhouse for cell culture.

The ratio of thrombin, calcium chloride solution and fibrinogen is variable from 4-6: 0.5-1, 5: 1 -3, preferably from 3: 1: 2.

The concentration of the calcium chloride solution is preferably 40umol / ml.

Stem cells used in step (b) can be selected from adipose tissue, umbilical cord blood and bone marrow mesenchymals. Stem cell concentration varies between 1x10 ⁵ and 2x10 ⁶ , preferably 1x10 ⁶ .

Culture medium can be selected from group 199 medium, MEM medium, RPMI medium, Dulbecco MEM medium, HAM F-12 nutrient, Leibowitz L-15 medium, HAM F-10 nutrient, SOL. BAI. From Hanks, half Alpha MEM, half Glasgow, half DMEM / HAM F-12, half RPMI 1640 with Hepes, half DMEM, half Schneider's, half TC-100, CTS, MesenCult SF Culture Kit and MesenCult XF Medium.

Step (e) is dispensed with if the selected culture medium is

CTS, MesenCult SF Culture Kit and MesenCult XF Medium. The serum may be selected from the group of inactivated fetal bovine serum (10-bio100i) (FBS), fetal bovine serum (10-bio100i) (FBS), sterile equine serum, calf serum (NCS) and human serum. In the exemplary embodiment of the present invention 4-0 Poly Vicryl J-304 Polyiglactin 910 Ethicon® suture sutures were used. The wires were opened into the laminar flow, then cut to a size of approximately 30 cm and placed in petri dishes (1 wire per plate). The fibrin glue used was prepared within the laminar flow with the following proportions: 20uL of fibrinogen, 30uL of thrombin and 10ul of calcium chloride.

The effect of glue formation is very rapid, so to facilitate handling we add 30uL of thrombin with 10uL of calcium chloride and only after all the wire preparation and cells dripping on the wire we add the 20uL of fibrinogen to finalize the procedure.

After 24 hours the plates containing adhered cell wires were observed under confocal microscopy to confirm the presence of cells.

To confirm the presence of cells adhered to the surface of the wire and between the filaments of the wire, confocal immunofluorescence microscopy and scanning electron microscopy were performed. In confocal immunofluorescence microscopy, the technique used was Live and Dead ^® .

The suture kit comprises the following components:

- Suture thread;

- thrombin and calcium chloride;

- fibrinogen;

- culture medium, and

- serum.

The ratio of thrombin, calcium chloride solution and fibrinogen is variable from 4-6: 0.5-1, 5: 1 -3, preferably from 3: 1: 2. The concentration of the calcium chloride solution is preferably 40umol / ml.

The serum may be selected from the group of inactivated fetal bovine serum (10-bio100i) (FBS), fetal bovine serum (10-bio100i) (FBS), sterile equine serum, calf serum (NCS) and human serum.

Stem cells is an optional component of the kit.

In vivo testing

A comparative in vivo study was performed with 3 groups of rats. Group 1 (control) had 5 cecal fistula animals without any treatment and were observed for 21 days. Group 2 (cell injection) had 8 animals with caecal fistula, received injection of 1 x ^{10 6} mesenchymal stem cells of adipose tissue around the cecal fistula and were observed for 21 days. Group 3 (suture of the present invention) had 9 cecal fistula animals, had the cecal fistula sutured with the suture of the present invention containing 1 x ^{10 6} adhered adipose mesenchymal stem cells, and were observed for 21 days.

Photos were taken on days 3, 6, 9, 12, 15, 17, 19 and 21. The ImageJ program (Image Processing and Analysis in Java) was used to survey the fistula areas of each animal.

The average healing and closure of the fistula from the point of view of the fistulae was 46.54% in group 1, 71, 80% in group 2 and 90.34% in group 3. Results showed that the recovery of animals that the present invention was applied (group 3) was more effective than the other groups.

Examples Cell adhesion was tested on other types of sutures and without the aid of fibrin glue.

In all types of yarn cell adhesion was similar. The choice for the 4-0 Poly Vicryl J-304 Polyiglactin 910 Ethicon® occurred because this type of suture is already widely used in surgery and its use has been released by Anvisa for many years.

Adherence of cells to the sutures without fibrin glue was not effective, as the manipulation of the thread caused a large amount of cells to unglue the thread. Fibrin glue caused these cells to adhere to the wire in large quantities and caused no effective cell loss during handling.

Mesenchymal stem cells are cells capable of self-renewal and differentiation in several cell lines. Recent advances in the use of stem cells have provided a new modality within medicine, which is the use of stem cells in cell therapy.

Stem cells were obtained from fat from patients who underwent the liposuction procedure.

Table 1: Control group - Evolution of fistula area and average percentage of reduction of areas. The control animals did not have any support.

Table 2: Cell Injection Group - Evolution of fistula area and average percentage of reduction of areas. Animals in the cell injection group received an injection of 1x10 ⁶ mesenchymal stem cells around the fistula.

Table 3: Suture thread group of the present invention - Evolution of the fistula area and mean percentage reduction of the areas. The animals in this group received a suture containing 1 x ^{10 6} mesenchymal stem cells.

Claims

1. Process for obtaining an enriched suture comprising the following steps:

(a) thrombin and calcium chloride drip along the entire length of the suture;

(b) stem cell dripping;

c) Fibrinogen drip;

d) Addition of culture medium sufficient to cover the suture;

e) Addition of 10% serum to the amount of culture medium added, and

Process according to Claim 1, characterized in that the ratio of thrombin, calcium chloride and fibrinogen varies between 4

6: 0.5-1.5: 1-3: preferably 3: 1: 2.

Process according to Claim 1, characterized in that the concentration of the calcium chloride solution is preferably 40umol / ml.

Process according to Claim 1, characterized in that the stem cells can be selected from mesenchymal adipose tissue, umbilical cord blood and bone marrow.

5. Process according to claim 1 characterized in that the concentration of stem cells is variable between 1 x 10 ⁵ and 2x10 ^6, preferably 1 x10 ^6.

Process according to Claim 1, characterized in that the culture medium may be selected from the group 199 medium, MEM medium, RPMI medium, Dulbecco MEM medium, HAM F-12 nutrient, Leibowitz L-15 medium, HAM F nutrient. -10, SOL. BAI. From Hanks, Alpha MEM medium, Glasgow Medium, DMEM / HAM F-12 medium, RPMI 1640 medium with Hepes, DMEM medium, Schneider's medium, TC-100 medium, CTS, MesenCult SF Culture Kit and MesenCult XF Medium.

Process according to Claim 1, characterized in that step (e) is dispensed with if the selected culture medium is CTS, MesenCult SF Culture Kit and MesenCult XF Medium.

Process according to Claim 1, characterized in that the serum may be selected from the group of inactivated fetal bovine serum (10-bio100i) (FBS), fetal bovine serum (10-bio100) (FBS), sterile equine serum, serum calf (NCS) and human serum.

Suture thread characterized in that it is enriched according to claims 1 to 8.

Use of suture as described in claim 9, characterized in that it is applicable to sutures of any nature.

Suture kit comprising the following components:

- Suture thread;

- thrombin and calcium chloride;

- fibrinogen;

- culture medium, and

- serum.

Kit according to Claim 11, characterized in that the ratio of thrombin, calcium chloride and fibrinogen varies between 4-6: 0.5-1, 5: 1 - 3, preferably 3: 1: 2.

A kit according to claim 11 characterized in that the concentration of the calcium chloride solution is preferably 40umol / ml.

Kit according to Claim 11, characterized in that the culture medium can be selected from the group medium 199, medium MEM, medium RPMI, medium

Dulbecco MEM, HAM F-12 nutrient, Leibowitz L-15 medium, HAM F-10 nutrient, SOL. BAI. From Hanks, half Alpha MEM, half Glasgow, half DMEM / HAM F-12, half RPMI 1640 with Hepes, half DMEM, half Schneider's, half TC-100, CTS, MesenCult SF Culture Kit and MesenCult XF Medium.

Kit according to claim 11, characterized in that the serum may be selected from the group of inactivated fetal bovine serum (10-bio100i) (FBS), fetal bovine serum (10-bio100) (FBS), sterile equine serum, calf serum (NCS) and human serum.

Kit according to Claim 11, characterized in that it is an optional stem cell component.

Use of the suture kit as described in claims 11 to 16, characterized in that it is applicable to sutures of any nature.