CN101066472B - Tissue engineered peripheral nerve graft and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biological tissue engineering, in particular to a tissue-engineered peripheral nerve for repairing nerve defect transplantation and a preparation method thereof. In the tissue-engineered peripheral nerve graft provided by the present invention, the seed cells are mixed cells of neuron-like cells and Schwann-like cells induced to differentiate by hair follicle neural crest stem cells (NCSC), and the scaffolds are based on oligopeptides CDPGYIGSR and CQAASIKVAV modified xenogeneic decellularized neural basement membrane tubes. The present invention also provides a preparation method of the tissue engineered peripheral nerve graft. The seed cells in the present invention, especially the neuron-like cells, have a better effect on promoting nerve regeneration, and provide clinical materials with a wide range of sources, no immunogenicity, no ethical issues involved, and better effects in repairing long-segment nerve defects tissue engineered neural grafts.

Description

Tissue engineered peripheral nerve graft and preparation method thereof

technical field

The invention relates to the technical field of biological tissue engineering, in particular to a tissue-engineered peripheral nerve for repairing nerve defect transplantation and a preparation method thereof.

Background technique

The basic biological characteristics of peripheral nerve regeneration are: after peripheral nerve injury, Wallerian degeneration occurs in the distal nerve, and the axon sprouts from the proximal axons slide forward in the gap between mesenchymal cells and grow to the distal Büngner zone. and grow to the target organ to form synaptic connections; Schwann cells myelinate the axons; restore the nerve structure and complete the process of nerve regeneration.

At present, tissue-engineered peripheral nerves at home and abroad are designed based on the basic biological characteristics of peripheral nerve regeneration. They have a specific three-dimensional structure scaffold-nerve conduit, which can accept the growth of regenerated axons and play a role in mechanically guiding the axons; the planted Schwann cells are distributed in an orderly manner in the scaffold, similar to Büngner's belt; Schwann cells have biological activity, Can secrete neurotrophic factors, create a microenvironment for axonal growth, support and guide axonal regeneration. However, the tissue-engineered nerves constructed under this idea are limited by nerve chemotaxis and contact guidance, and the bridging distance generally does not exceed 30 mm. The main problem in clinical practice is that the source of autologous nerves is insufficient in long-segment defects; Axon regeneration needs to span two anastomoses, which increases the difficulty of nerve regeneration; in addition, the growth time of new axons is too long and they enter by mistake during the growth process, which is prone to disuse atrophy of effector or nerve-effector Mismatch etc. These problems restrict the clinical application of tissue engineered nerves.

In 2003, Stephen et al. transplanted neural stem cells isolated from the spinal cord to the distal stump of the transected rat sciatic nerve, and found that the neural stem cells transformed into motor neuron-like cells, and extended axons into muscles to form cholinergic synapses. Inspired by this study, in order to solve the deficiencies of the current tissue-engineered nerves, the present invention uses neurons as the main cells to construct tissue-engineered peripheral nerves, and uses the idea and method of bridging neurons to reconstruct the defective nerve function, which can overcome the inductive The bridging distance of tissue-engineered nerves is short, axon regeneration needs to span two anastomoses and the growth time of new axons is too long.

The key factor in constructing tissue engineering is seed cells. Currently, the seed cells that have been studied more in tissue engineering neural construction are Schwann cells, bone marrow mesenchymal stem cells, and neural stem cells. Autologous peripheral Schwann cells are end-stage cells with poor proliferative ability, difficulty in in vitro isolation and culture, decreased activity, and the need for a second operation. Although the immortal strain of Schwann cells has been obtained through genetic modification, its secretion is conducive to nerve repair. The ability of factor is also significantly decreased; allograft often faces immune rejection. Bone marrow mesenchymal stem cells have the advantages of convenient extraction, rapid expansion, and avoidance of immune rejection caused by allogeneic nerve transplantation, but the rate of induction into neuron-like cells is relatively low. Neural stem cells, as new seed cells for tissue engineered nerves, have been a research hotspot in recent years, but the source of autologous neural stem cells is difficult, and allogeneic neural stem cells also have the problem of immune rejection. Therefore, finding new seed cells should be the top priority for tissue engineering neural construction.

The research of Sieber-Blum et al. showed that there are pluripotent stem cells in the adult hair follicle bulge, and they are derived from the neural crest, so they are named hair follicle neural crest stem cells (NCSC), which can naturally differentiate into neurons and smooth muscles. cells, SC and melanocytes, etc. Amoh et al. found that stem cells in the hair follicle bulge were positive for Nestin and could form neurons. After being induced and differentiated by 10% fetal bovine serum, nearly half of the cells differentiated into neurons (48%), and 30% of the cells differentiated into neurons. Cells differentiated into glial cells, and other cells such as keratinocytes (18%), smooth muscle cells (2%), melanocytes (2%), etc., and these Nestin-positive hair follicle stem cells were transplanted into mouse sciatic nerve defects It can make it transdifferentiate into Schwann cells, and play a good role in repairing nerve defects. It can be seen that hair follicle NCSC can be derived from itself, which is convenient to obtain, has little damage, does not involve immunogenicity and ethical issues, and has the advantage of differentiating into nerve cells.

Scaffold materials are another key to determine whether tissue engineered nerves can be successfully constructed and applied clinically. Yu et al. showed that laminin (see Biomaterials, 2005, 26(13): 1507-1514.) is an extracellular matrix that can enhance cell adhesion, migration, differentiation and gene expression, and the YIGSR sequence located in its β1 chain It can enhance the adhesion of nerve cells. The IKVAV sequence located on the A chain can promote the axon growth of nerve cells. The extended amino acid sequence CDPGYIGSR and CQAASIKVAV can further enhance the adhesion of cells and promote the growth of axons.

Contents of the invention

The object of the present invention is to provide a tissue-engineered peripheral nerve graft that can repair long-segment nerve defects, and a preparation method thereof, with wide material sources, strong activity, no immunogenicity, no ethical issues involved, and the ability to repair long-segment nerve defects.

The present invention provides a tissue engineered peripheral nerve graft, comprising seed cells and scaffolds, wherein the seed cells are mixed cells of neuron-like cells and Schwann-like cells induced to differentiate from follicular neural crest stem cells (NCSC), wherein The scaffold is a heterogeneous decellularized neural basement membrane tube modified with oligopeptides having the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The invention provides a preparation method of tissue-engineered peripheral nerve grafts, including the separation, cultivation, purification, expansion and identification of hair follicle neural crest stem cells (NCSC); the induction and differentiation of hair follicle NCSC to neuron-like and Schwann-like cells and identification; preparation and modification of xenogeneic decellularized nerve basement membrane tube scaffolds; in vitro construction of neuronal tissue engineered peripheral nerves.

Specific steps are as follows:

1. Preparation of adult hair follicle neural crest stem cells (NCSC)

Hair follicle NCSCs were isolated from adult hair follicle bulges by the patch method (see Sieber-Blum et al., Developmental Dynamics, 2004, 231: 258-269.). The skin hair follicle bulge of the vibrissa pad of clean SD rats was taken, cultured as a block in neural stem cell culture medium, and subcultured to obtain hair follicle NCSC.

2. Induction and differentiation of adult hair follicle NCSC

Retinoic acid RA (5-20 μmol/L; purchased from Sigma) and hedgehog agonist (hedgehogagonist, HhAg1.3, 5-20 μmol/L; purchased from Curis Inc.) were combined to induce hair follicle NCSC to differentiate into neuron-like cell. The best induction concentration is 10μmol/L. The action time is 1d to 14d, and the best action time is 7d.

Hair follicle NCSCs were induced to differentiate into Schwann-like cells with neuregulin-1 (NRG1, 50-150 ng/ml, purchased from Sigma). The best induction concentration is 100ng/ml. The action time is 1d to 14d, and the best action time is 7d.

3. Preparation and Modification of Xenogeneic Decellularized Basement Membrane Tube Scaffolds

The canine sciatic nerve was treated with 1% lysolecithin, etc. to obtain the heterogeneous decellularized nerve basement membrane tube scaffold. And the oligopeptide CDPGYIGSR (SEQ ID NO: 1), CQAASIKVAV (SEQ ID NO: 2) was combined to soak the scaffold in a ratio of 1:1, the optimal soaking time was 12 hours, lyophilized, ethylene oxide sterilized, - Store at 20°C for later use.

4. Construction of neuronal tissue engineered peripheral nerves in vitro

Under the operating microscope, neuron-like cells and Schwann-like cells induced and differentiated by hair follicle NCSC were planted at a ratio of 1:1, 2:1, and 3:1, respectively, and the heterogeneous decellularized neural basement membrane tube scaffold compounded with seed cells was placed. In vitro construction was carried out in a rotating culture chamber.

The seed cells of the present invention are selected from hair follicle neural crest stem cells (NCSC), which can not only be derived from themselves, but also have a wide range of sources, convenient material collection, small damage, strong activity, and no immunogenicity and ethical issues. NCSC-induced neuron-like Cells have a better promoting effect on nerve regeneration; in the present invention, the oligopeptide CDPGYIGSR and CQAASIKVAV are combined to modify the scaffold material, which is more conducive to the adhesion, migration and differentiation of seed cells.

Detailed ways

Now, the present invention will be further described in conjunction with the embodiments, but the implementation of the present invention is not limited thereto.

Example 1:

1. Preparation of adult hair follicle neural crest stem cells (NCSC)

(1) Primary culture of hair follicle NCSC

Clean grade SD rats, weighing 100 g, were sacrificed by cervical dislocation. Disinfect with 75% ethanol, take rat vibrissa pad hair follicles, at room temperature, under a microscope, under sterile conditions, take 10-20 hair follicles from each rat, for preparing 1-2 tissue-engineered nerves. Put the complete hair follicle in 4°C DPBS, cut off the hair follicle bulb, peel off the outer connective tissue of the hair follicle, and expose the hair follicle bulge (keep the hair shaft 1mm, which is convenient for block operation), wash 2 times with 4°C DPBS, and set aside . 50 μl of rat tail gum (1 mg/ml, purchased from Sigma) was evenly coated on each 35 mm culture dish, dried on a clean bench at room temperature for more than 1 h, and incubated with the primary culture medium in a 37° C., 5% CO2 incubator for 3 h for later use. Plant 5-8 hair follicle protuberances per 35mm culture dish, let it stand in a 37°C, 5% CO2 incubator for 1 hour, and then slowly add the primary culture medium [DMEM/F12 (1:1), purchased from GIBCO; B27 (2%), purchased from GIBCO; N2 (1%), purchased from GIBCO; ITS+3 (insulin, transferrin, selenium, and three essential fatty acids), purchased from Sigma; bFGF (20ng/ml), EGF (20ng/ ml); L-glutamine (200 μmol/L); fetal bovine serum (10%), purchased from GIBCO], cultured statically for 4 to 6 days and then subcultured.

(2) Subculture of hair follicle NCSC

Remove hair follicle protrusions with microscopic instruments, digest with 0.25% trypsin for 2 minutes, stop digestion with trypsin inhibitor, add 2ml of culture medium (without fetal bovine serum, the rest of the ingredients are the same as the original culture medium) per 35mm dish, and pipette to prepare Single cell suspension, the cell density is 1×10 ⁵ /ml. Culture in a 37°C, 5% CO ₂ incubator, and change half of the solution every two days.

2. Induction and differentiation of adult hair follicle NCSC

(1) Induced differentiation into neuron-like cells

The second-generation hair follicle NCSCs were taken, and 10 μmol/L retinoic acid RA (Sigma) and 10 μmol/L hedgehog agonist (hedgehog agonist, HhAg1.3, Curis Inc.) were added for 7 days.

(2) Induced differentiation to Schwann-like cells

The second-generation hair follicle NCSCs were taken and differentiated with 100ng/ml neuregulin-1 (NRG1, Sigma) for 7 days, after which Schwann-like cells were identified.

3. Preparation and Modification of Xenogeneic Decellularized Basement Membrane Tube Scaffolds

Entrust Shanghai Sangon Bioengineering Co., Ltd. to synthesize oligopeptides CDPGYIGSR (SEQ ID NO: 1) and CQAASIKVAV (SEQ ID NO: 2).

Canine sciatic nerve was collected under sterile conditions, DPBS (pH 7.3) at 4°C for 5 days, 1% lysolecithin/0.1mol/L PBS (pH 7.0), at room temperature for 4 days, the medium was changed once every 24 hours, and washed with DPBS for 30 minutes. DNase I (600U/ml), RNase A (10U/ml), 37°C, 24h, washed 3 times with DPBS, 1h, oligopeptide CDPGYIGSR/CQAASIKVAV (1:1, 1.5mg/ml, dissolved in PBS) soaked for 12h , during which there is a slight vibration. Rinse thoroughly with PBS three times, lyophilize at low pressure, sterilize with ethylene oxide, and store at -20°C for later use.

4. Construction of neuronal tissue engineered peripheral nerves in vitro

The planting density of the seed cells is 1×10 ⁶ /ml～1×10 ⁷ /ml, and under the operating microscope, the neuron-like cells induced by hair follicle NCSC are planted at the ratio of 1:1, 2:1, and 3:1, respectively. and Schwann-like cells.

Microinject first (inject the mixed suspension of seed cells into the scaffold with a micro-sampler under a microscopic surgical microscope, with an interval of 5 mm, and inject 20-50 μl each time), and then infiltrate and inoculate (the mixed suspension of seed cells is evenly added dropwise) On the support, incubate in the culture dish in suspension for 4 hours), and then rotate the culture (fix the support in the rotating culture chamber and cultivate for 1-2 days to make the cell distribution more uniform). After 2 days, it was used for transplantation and repair of sciatic nerve defect in SD rats.

Example 2:

1. Repair of sciatic nerve defect in SD rats with tissue engineered peripheral nerve graft

Cut off 10mm of sciatic nerve on one side of adult male SD rats (the skin of which has been taken for isolation and culture of hair follicle NCSC), and keep the distance between the stumps at 15mm, and use microsurgical techniques to connect the nerve graft to the stump of sciatic nerve; Operation. Animals survived for 9 weeks.

Animals were divided into groups as follows: autologous nerve transplantation group, tissue engineered peripheral nerve transplantation group (hair follicle NCSC-induced neuron-like cells and Schwann-like cells were implanted at a ratio of 1:1, 2:1, and 3:1, respectively), Inducible tissue engineered nerve transplantation group (only Schwann-like cells induced by hair follicle NCSC implantation), canine decellularized nerve basement membrane tube scaffold transplantation group. There were 5 animals in each group. The sciatic nerve excised in the autologous nerve transplantation group was 15mm long, and this segment of nerve was orthotopically transplanted to repair the defective nerve.

2. Evaluation of Functional Recovery

(1) According to the de Medinaceli method (see Exp Neurol, 1982, 77:634), measure the rat hind paw imprint, and calculate the sciatic nerve function index.

(2) The number of times the animals in each group touched the plate was detected with a flat step trapezoidal instrument to evaluate their functional recovery. The fewer touches, the better the recovery of function.

(3) After 9 weeks of operation, electrical stimulation was performed at the proximal end of the regenerated nerve, and the action potential was recorded at the distal end, while the contraction of the gastrocnemius muscle was observed; the weight and muscle strength of the triceps calf were measured.

The study found that the functional recovery of the tissue-engineered peripheral nerve transplantation group was better than that of the induced tissue-engineered nerve transplantation group and the empty scaffold transplantation group, and was similar to that of the autologous nerve transplantation group. Among them, the experimental group with the best effect was the implantation group of hair follicle NCSC-induced neuron-like cells and Schwann-like cells at a ratio of 1:1.

SEQUENCE LISTING

<110> The Second Military Medical University of the Chinese People's Liberation Army

<120> Tissue engineered peripheral nerve graft and preparation method thereof

<130> specification, claims

<160>2

<170>PatentIn version 3.1

<210>1

<211>9

<212>PRT

<213> Artificial sequence

<400>1

Cys Asp Pro Gly Tyr Ile Gly Ser Arg

1 5

<210>2

<211>10

<212>PRT

<213> Artificial sequence

<400>2

Cys Gln Ala Ala Ser Ile Lys Val Ala Val

1 5 10

Claims (5)

1. A tissue engineered peripheral nerve graft, comprising seed cells and a scaffold, wherein the seed cells are mixed cells of neuron-like cells and Schwann-like cells induced by hair follicle neural crest stem cells, wherein the scaffold It is a heterogeneous decellularized nerve basement membrane tube modified with oligopeptides having the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. 2. A tissue-engineered peripheral nerve graft according to claim 1, wherein the ratio of neuron-like cells to Schwann-like cells is 1:1, 2:1 or 3:1. 3. a preparation method of tissue engineering peripheral nerve graft as claimed in claim 1, is characterized in that it comprises the following steps: A) Isolation, cultivation, purification, expansion and identification of hair follicle neural crest stem cells; B) Induction and identification of hair follicle neural crest stem cells to neuron-like cells and Schwann-like cells: the induction and differentiation conditions of hair follicle neural crest stem cells to neuron-like cells are 5-20 μmol/L retinoic acid and 5-20 μmol/L L's hedgehog agonist combined with induction of hair follicle neural crest stem cells, the action time is 1d ~ 14d; hair follicle neural crest stem cells are induced to differentiate into Schwann cell-like cells with 50 ~ 150ng/ml neuregulin to induce hair follicle neural crest stem cells, the effect The time is 1d～14d; C) Preparation of heterogeneous decellularized nerve basement membrane tube scaffold and modification with oligopeptides having the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in combination with soaking scaffold in a ratio of 1:1; D) In vitro construction of tissue-engineered peripheral nerve grafts: First, a mixture of neuron-like cells and Schwann-like cells induced by hair follicle neural crest stem cells was planted on the scaffold, and then the scaffold was placed in a rotating culture chamber for in vitro Construct. 4. the preparation method of a kind of tissue engineered peripheral nerve graft according to claim 3, is characterized in that in wherein B) step, the induction differentiation condition of hair follicle neural crest stem cell to neuron-like cell is 10 μ mol/L The combination of retinoic acid and 10 μmol/L hedgehog agonist induced hair follicle neural crest stem cells for 7 days; the condition for inducing the differentiation of hair follicle neural crest stem cells to Schwann-like cells was to induce hair follicle neural crest stem cells with 100ng/ml neuregulin , The action time is 7d. 5. the preparation method of a kind of tissue engineered peripheral nerve graft according to claim 3, is characterized in that in wherein C) step, with SEQ ID NO:1 and the aminoacid sequence shown in SEQ ID NO:2 The oligopeptides were modified in combination with soaking supports at a ratio of 1:1, and the soaking time was 12 hours, with slight vibration during soaking.