CN113332495B

CN113332495B - Three-dimensional vascularized tissue engineering bone and preparation method thereof

Info

Publication number: CN113332495B
Application number: CN202110601076.XA
Authority: CN
Inventors: 叶鑫; 俞梦飞; 游东奇; 陆科杰; 刘超; 王少龙; 叶冠琛
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2023-03-21
Anticipated expiration: 2041-05-31
Also published as: CN113332495A

Abstract

The invention discloses a three-dimensional vascularized tissue engineering bone, which is characterized in that: the scaffold comprises a scaffold, mesenchymal stem cells and vascular endothelial cells, wherein the mesenchymal stem cells and the vascular endothelial cells are dispersed in the scaffold. Compared with the background art, the invention has the beneficial effects that: the three-dimensional vascularized tissue engineering bone manufactured by the invention can realize that the directional migration of vascular endothelial cells and mesenchymal stem cells is regulated and controlled by the gradient hydrogel, a three-dimensional vascular perfusion network similar to autologous jaw bone tissue is formed, the blood supply condition of the tissue engineering bone is improved, the tissue engineering bone is suitable for the transplantation reconstruction of jaw bone defects with poor blood supply condition, the survival of the tissue engineering bone is ensured, the success rate of jaw bone defect reconstruction and repair is favorably improved, the problem of secondary damage of a supply area caused by the current clinical fibula flap is avoided, the damage and the operation area of a patient are reduced, and the minimally invasive and efficient jaw bone reconstruction is favorably realized.

Description

Three-dimensional vascularized tissue engineering bone and preparation method thereof

Technical Field

The invention belongs to the technical field of oral medical instruments, and particularly relates to a three-dimensional vascularized tissue engineering bone and a preparation method thereof.

Background

The repair of defects in jaw bone tissue due to tumors, trauma, inflammation, and the like has been a significant challenge for oromaxillofacial surgeons. The mandible, which is the only movable bone in the maxillofacial bone, plays an important role in maintaining the maxillofacial appearance, and also plays important roles in chewing, speech, and the like. Functional reconstructive repair of defects in jaw bone tissue has therefore been an important issue for regenerative medicine. The current clinical gold standard for repairing large-area defects of jawbones is still the self fibula skin flap transplantation of patients, but the fibula flap inevitably causes secondary operation damage of a calf supply area in the preparation process, the long and thin shape of the fibula is different from the irregular shape of the jawbones, the ideal shape effect is difficult to obtain after reconstruction, the stress distribution is uneven when the fibula is subjected to chewing force, and the bone absorption is caused, so that the jaw reconstruction failure is caused. The tissue engineering bone is used as a bone grafting substitute material, can be used for replacing autologous bone for grafting and repairing bone defects, and can avoid secondary operation trauma to patients.

However, the development of tissue engineering bone aiming at jaw defects is less at present, because compared with long bones such as thighbone and the like, the reconstruction and repair of the long bones are difficult greatly due to the unique anatomical structure and physiological characteristics of the mandible, because the bone density of the mandible is more compact and the blood vessel supply is single compared with the long bones, the bone density of the mandible is mainly supplied by the inferior alveolar artery positioned in the mandible, the collateral circulation is less, and when the jaw defects are repaired by simply using autologous free bone blocks or bone grafting artificial materials, insufficient blood supply and ischemic necrosis are easy to occur to cause the repair failure. Thus, the repair of jaw defects places higher demands on the blood supply conditions of tissue engineered bones.

Disclosure of Invention

The present invention overcomes at least one of the deficiencies of the prior art described above,

the invention aims to provide a three-dimensional vascularized tissue engineering bone which comprises a stent, mesenchymal stem cells and vascular endothelial cells, wherein the mesenchymal stem cells and the vascular endothelial cells are dispersed in the stent.

Further, the hydrogel fills pores of the stent, and the mesenchymal stem cells and the vascular endothelial cells are dispersed in the hydrogel.

Further, the pores of the scaffold are filled with hydrogels of different concentrations.

Furthermore, the hydrogel with different concentrations comprises 1-2% of matrigel and 5% of GelMA powder by mass, the matrigel and the GelMA powder are premixed and then added with physiological saline to form a gradient hydrogel premix solution with a concentration gradient of 1-2%, and the gradient hydrogel premix solution is injected into a stent and is cured by ultraviolet light.

Further, the cell density of the mesenchymal stem cells and the vascular endothelial cells is 200 ten thousand/ml.

Furthermore, one end of the bracket is hydrogel containing 1% of matrigel by mass, and the other end of the bracket is hydrogel containing 2% of matrigel by mass; the hydrogel with gradually changed matrigel concentration is arranged in the middle. The GelMA powder and the balance of normal saline are respectively contained in 5 percent by mass.

Furthermore, one end of the bracket is hydrogel containing 1 mass percent of matrigel, and the other end of the bracket is hydrogel containing 2 mass percent of matrix. The GelMA powder and the balance of normal saline are respectively contained in 5 percent by mass.

The invention also aims to provide a preparation method of the three-dimensional vascularized tissue engineering bone, which comprises the following steps:

s1, printing support

S2, pouring 1% concentration cell-loaded hydrogel premix solution on the support, and carrying out ultraviolet light curing to obtain a low-concentration gradient cell-loaded hydrogel module;

and S3, pouring the cell-loaded hydrogel premix solution with the concentration of 2% on the support, and carrying out ultraviolet light curing to obtain a high-concentration gradient cell-loaded hydrogel module and obtain the three-dimensional vascularized tissue engineering bone.

Further, a part 1/2 below the bracket is a low-concentration gradient cell-loaded hydrogel module; the upper 1/2 part of the bracket is a high concentration gradient cell-loaded hydrogel module. The low-concentration blood vessel firstly contacts with the human body and contacts with the mandibular artery of the human body, and then the gradient difference is formed from the low concentration to the high concentration, and the blood vessel grows upwards.

Furthermore, before ultraviolet curing, the hydrogel premix needs to be resuspended with mesenchymal stem cells and vascular endothelial cells.

Compared with the background art, the invention has the beneficial effects that:

(1) The three-dimensional vascularized tissue engineering bone manufactured by the invention can realize that the directional migration of vascular endothelial cells and mesenchymal stem cells (directional migration from a low-concentration hydrogel module to a high-concentration hydrogel module) is regulated and controlled by gradient hydrogel, a three-dimensional vascular perfusion network similar to autologous jaw bone tissue is formed, the blood supply condition of the tissue engineering bone is improved, the tissue engineering bone is suitable for transplantation reconstruction of jaw bone defects with poor blood supply condition, the survival of the tissue engineering bone is ensured, the success rate of jaw bone defect reconstruction and repair is favorably improved, the problem of secondary damage of a supply area caused by the current clinical fibula flap is avoided, the damage and the operation area of a patient are reduced, and the minimally invasive and efficient jaw bone reconstruction is favorably realized.

(2) The three-dimensional vascularization tissue engineering bone is completely matched with the defective jaw bone of the affected side of the patient, and can accurately recover the original appearance and chewing function of the patient.

Drawings

FIG. 1 is a schematic diagram of a three-dimensional vascularized tissue-engineered bone structure according to the present invention.

FIG. 2 is a schematic diagram of preparation of a low concentration gradient cell-loaded hydrogel based on a scaffold.

Fig. 3 is a schematic diagram of a high concentration gradient hydrogel module prepared based on a stent and a low concentration gradient hydrogel module.

Fig. 4 is a schematic diagram of the three-dimensional vascularized tissue engineering bone used for large-scale jaw defect repair and reconstruction.

Fig. 5 is an enlarged view of a three-dimensional vascularized tissue-engineered bone.

Fig. 6 is a schematic diagram of a capillary network that can be formed in three dimensions in vitro.

Fig. 7 is a schematic diagram of a vessel lumen structure with real blood vessels.

Fig. 8 is a schematic diagram of a three-dimensional vascularized tissue engineering bone used for repairing and reconstructing a defect of a small range jaw bone.

Wherein, 1 is a stent, 2 is low concentration gradient hydrogel, 3 is high concentration gradient hydrogel, 4 is vascular endothelial cells, 5 is mesenchymal stem cells, 6 is a three-dimensional vascular network, 7 is a capillary vascular network structure, and 8 is a vascular lumen structure.

Detailed Description

The drawings are for illustrative purposes only and are not to be construed as limiting the patent; it will be understood by those skilled in the art that certain well-known structures and descriptions thereof may be omitted. All the medicines are commercially available products unless otherwise noted.

The invention relates to a three-dimensional vascularized tissue engineering bone, which comprises a scaffold, hydrogel, mesenchymal stem cells and vascular endothelial cells, wherein the mesenchymal stem cells and the vascular endothelial cells are dispersed in the hydrogel, and the hydrogel fills pores of the scaffold.

The fiber diameter of the scaffold is 150-250 microns, and the pores of the scaffold are 200-400 microns. The gradient hydrogel comprises 1-2% of matrigel and 5% of GelMA powder in percentage by mass, the matrigel and the GelMA powder are premixed and then added with normal saline to form a gradient hydrogel premix solution with the concentration gradient of 1% and 2%, and the gradient hydrogel premix solution, mesenchymal stem cells with the cell density of 200 ten thousand/ml and vascular endothelial cells are re-suspended and then injected into a stent to be cured by ultraviolet light. The bracket is designed according to the shape of a jaw bone on a healthy side and is manufactured by polyion compound materials through an extrusion type three-dimensional printing technology.

( Such as F.Zhu, L.Cheng, J.yin, Z.L.Wu, J.Qian, J.Fu, Q.ZHEN, 3 DPringing of Ultratough polymerization hydrocarbons, ACS applied Mater Interfaces8 (45) (2016) 31304-31310. A method for preparing high strength and high toughness Polyion hydrogel scaffolds by 3D printing. The patent number is as follows: ZL201610634276.4 )

The invention relates to a preparation method of a three-dimensional vascularized tissue engineering bone, which comprises the following steps:

s1, printing a support;

s2, pouring the cell-loaded hydrogel premix solution with the concentration of 1% into a 1/2 part below the bracket, and carrying out ultraviolet curing to obtain a low-concentration gradient cell-loaded hydrogel module;

and S3, pouring the cell-loaded hydrogel premix solution with the concentration of 2% on 1/2 part of the scaffold, and carrying out ultraviolet curing to obtain a high-concentration gradient cell-loaded hydrogel module and obtain the three-dimensional vascularized tissue engineering bone.

Further, before step S1, the CT scan image of the jaw bone of the patient needs to be processed, the healthy jaw bone data is used to mirror the jaw bone defect model of the affected side, and the design of the three-dimensional vascularized tissue engineering bone scaffold is performed according to the jaw bone defect model.

( Extrusion Printing methods such as F.Zhu, L.Cheng, J.yin, Z.L.Wu, J.Qian, J.Fu, Q.ZHEN, 3D Printing of Ultratough Polymer hydrogels, ACS applied Mater Interfaces8 (45) (2016) 31304-31310A method for making high strength and high toughness Polyion hydrogel scaffolds using 3D Printing. The patent number is as follows: ZL201610634276.4 )

A three-dimensional vascularized tissue engineering bone comprises a basic scaffold for personalized restoration of jaw bone defects, a gradient hydrogel for filling scaffold pores and loading seed cells, vascular endothelial cells for vascular tissue regeneration and mesenchymal stem cells for bone tissue regeneration; the basic bracket is designed according to the jaw shape of a healthy side, is prepared from polyion compound (PIC) materials through an extrusion type three-dimensional printing technology, and is used for recovering the defective jaw shape and ensuring the mechanical strength of a transplanting material; the gradient hydrogel is formed by GelMA added with different Matrigel (Matrigel), and has the effects of regulating and controlling the formation of three-dimensional vascular network by vascular endothelial cells and promoting the osteogenic differentiation of mesenchymal stem cells. And the cell-loaded gradient hydrogel with different concentration gradients is filled in the PIC bracket in a layered manner to form a low-concentration gradient hydrogel module and a high-concentration gradient hydrogel module respectively, and the construction of the three-dimensional vascularized tissue engineering bone is finally completed after in vitro culture for one week.

The basic PIC stent shape of the three-dimensional vascularized tissue engineering bone is consistent with the jaw bone defect shape of a patient, the fiber diameter of the PIC stent is 150-250 microns, and the pores of the stent are 200-400 microns and are close to the cancellous bone pores of the human jaw bone. The gradient hydrogel filled in pores and loaded with seed cells in the three-dimensional vascularized tissue engineering bone scaffold has two concentrations, namely a low-concentration gradient hydrogel premix formed by adding 1% of matrigel and 5% of GelMA powder by mass into physiological saline with corresponding volume and a high-concentration gradient hydrogel premix formed by adding 2% of matrigel and 5% of GelMA powder by mass into physiological saline with corresponding volume. And injecting the premixed solution into the PIC stent in layers, curing by using ultraviolet light, and culturing in vitro for a week to form the three-dimensional vascularized tissue engineering bone with a three-dimensional vascularized structure and a good bone regeneration effect.

A preparation method of a three-dimensional vascularized tissue engineering bone comprises the following steps:

(1) Carry out CT scan to patient's jaw, handle CT image, utilize healthy side jaw data mirror image to rebuild the jaw of suffering from the side and fall the model to carry out jaw bionic tissue scaffold's design according to jaw falls the model

(2) PIC stent manufactured by extrusion three-dimensional printing method using polyionic compound

(3) The 1/2 part of the PIC stent is firstly filled with the low-concentration gradient hydrogel premix solution loaded with the mesenchymal stem cells and the vascular endothelial cells and then is cured by ultraviolet light to complete the construction of the low-concentration gradient hydrogel module

(4) Pouring the high-concentration gradient hydrogel premixed solution loaded with the mesenchymal stem cells and the vascular endothelial cells into 1/2 part of the PIC stent, and then carrying out ultraviolet curing to complete the construction of a high-concentration gradient hydrogel module

(5) After one week of in vitro culture, the construction of the three-dimensional vascularized tissue engineering bone is completed.

The construction of the gradient hydrogel module can regulate and control the directional migration of vascular endothelial cells and the angiogenesis processes of the sprouting, the formation of follicular cells, the formation of lumens and the like of the vascular endothelial cells, promote the construction of a three-dimensional vascular perfusion network in the tissue engineering bone, improve the blood supply condition of the tissue engineering bone, and is suitable for the repair and reconstruction of the jaw bone defect with single blood supply.

Example 1:

as shown in fig. 1, the present embodiment provides a three-dimensional vascularized tissue-engineered bone and a method for preparing the same. Comprises a basic scaffold 1 for repairing individualized jaw bones, a low-concentration gradient hydrogel 2 for filling scaffold pores and loading seed cells, a high-concentration gradient hydrogel 3 for filling scaffold pores and loading seed cells, vascular endothelial cells 4 for regenerating vascular tissues and mesenchymal stem cells 5 for regenerating bone tissues; the basic bracket is designed according to the jaw shape of a healthy side, is prepared from polyion compound (PIC) materials through an extrusion type three-dimensional printing technology, and is used for recovering the defective jaw shape and ensuring the mechanical strength of a transplanting material; the gradient hydrogel is formed by GelMA added with different Matrigel (Matrigel), and has the effects of regulating and controlling the formation of three-dimensional vascular network by vascular endothelial cells and promoting the osteogenic differentiation of mesenchymal stem cells. And the cell-loaded gradient hydrogel with different concentration gradients is filled in the PIC bracket in a layered manner to form a low-concentration gradient hydrogel module and a high-concentration gradient hydrogel module respectively, and the construction of the three-dimensional vascularized tissue engineering bone is finally completed after in vitro culture for one week.

Example 2:

a method for repairing a jaw defect using the three-dimensional vascularized tissue-engineered bone of example 1 is provided, which comprises the following steps:

the first step is as follows: carrying out CT scanning on jaw bones of a patient, processing CT images, and carrying out mirror image reconstruction on jaw bone defect models on affected sides by using healthy side jaw bone data;

the second step is that: printing a PIC stent 1 with the size consistent with the jaw defect size by using an extrusion printing method according to the jaw defect model;

the third step: pouring low-concentration gradient hydrogel 2 loaded with vascular endothelial cells 4 and mesenchymal stem cells 5 on the basis of the PIC stent 1, and completing the construction of a low-concentration gradient hydrogel module after ultraviolet light curing (figure 2);

the fourth step: pouring low-concentration gradient hydrogel 3 loaded with vascular endothelial cells 4 and mesenchymal stem cells 5 on the basis of the PIC stent 1 and the low-concentration gradient hydrogel module, and completing the construction of the high-concentration gradient hydrogel module after ultraviolet light curing (figure 3);

the fifth step: after one week in vitro culture of the PIC scaffold complex with low and high concentration hydrogel modules, a three-dimensional vascularized tissue engineered bone with a three-dimensional vascular perfusion network was formed (fig. 1).

Example 3:

the rabbit suffering from jaw bone defect uses the three-dimensional vascularization tissue engineering bone to repair large-area jaw bone defect as an example. Materials and apparatus

1.1 repair materials

GelMA (EFL, china), matrigel (Gibco, usa), PBS (Gibco, usa), PIC solution (oral hospital laboratory affiliated to the medical institute of zhejiang university, china), titanium powder (oral hospital laboratory affiliated to the medical institute of zhejiang university, china)

1.2 Main Equipment

FDM extrusion printer (Chinese, affiliated oral Hospital laboratory of Zhejiang university medical college), LED UV point light source machine (China, rinshixi electronic technology)

The first step is as follows: carrying out CT scanning on the jaw bone of a patient, processing a CT image, and carrying out mirror image reconstruction on a jaw bone defect model on the affected side by using healthy side jaw bone data;

the fifth step: after one week of in vitro culture of the PIC scaffold complex having the low concentration hydrogel module and the high concentration hydrogel module, a three-dimensional vascularized tissue engineering bone having a three-dimensional vascular perfusion network was formed (fig. 1) and transplanted to the patient's defective jawbone 5, completing the repair of the jawbone defect (fig. 4).

The three-dimensional vascularized tissue engineering bone can form a three-dimensional capillary network in vitro (figure 6, arrows show vascular endothelial cells which are mutually connected to form a capillary network-shaped structure, the number of vascular branches reaches 130.8/mm < 2 >, and the number of vascular branch nodes reaches 100.3/mm < 2 >), and has a real vascular lumen structure (figure 7, a laser confocal microscope can observe that a lumen-like follicle (lumen) structure is formed inside the vascular endothelial cells, and branches (branch) appear).

Example 4:

the method is used for repairing small-area jaw bone defects by using three-dimensional vascularized tissue engineering bone of the invention as an example. Materials and apparatus

1.1 repair materials

1.2 Main Equipment

the second step is that: printing a PIC bracket 1 with the size consistent with the jaw defect size by using an extrusion printing method according to the jaw defect model;

the third step: pouring low-concentration gradient hydrogel 2 loaded with vascular endothelial cells 4 and mesenchymal stem cells 5 on the basis of the PIC stent 1, and completing construction of a low-concentration gradient hydrogel module after ultraviolet light curing;

the fourth step: after the PIC stent complex with the pure low-concentration hydrogel module is cultured in vitro for one week, a three-dimensional vascularized tissue engineering bone with a three-dimensional vascular perfusion network is formed and transplanted to the defective jaw bone of a patient, and the repair of the jaw bone defect is completed (figure 8).

Claims

1. A three-dimensional vascularized tissue-engineered bone, characterized in that: the mesenchymal stem cells and the vascular endothelial cells are dispersed in the stent; further comprising a hydrogel filling the scaffold pores, the mesenchymal stem cells and vascular endothelial cells being dispersed within the hydrogel; the pores of the scaffold are filled with hydrogels of different concentrations; the hydrogel has two concentrations, namely a low-concentration gradient hydrogel premix formed by adding 1% of matrigel and 5% of GelMA powder by mass into corresponding volume of physiological saline and a high-concentration gradient hydrogel premix formed by adding 2% of matrigel and 5% of GelMA powder by mass into corresponding volume of physiological saline; after the premixed solution is injected into the scaffold layer by layer, the premixed solution is cured by ultraviolet light and then cultured in vitro for a week to form the three-dimensional vascularized tissue engineering bone with a three-dimensional vascularized structure and a good bone regeneration effect.

2. The three-dimensional vascularized tissue-engineered bone of claim 1, wherein: the cell density of the mesenchymal stem cells and the vascular endothelial cells is 200 ten thousand per milliliter.

3. A preparation method of a three-dimensional vascularized tissue engineering bone is characterized by comprising the following steps:

s1. Printing support

S2, pouring 1% hydrogel premix liquid on the support, and carrying out ultraviolet curing to obtain a low-concentration gradient hydrogel module;

and S3, pouring 2% hydrogel premix liquid on the support, and carrying out ultraviolet curing to obtain a high-concentration gradient hydrogel module so as to obtain the three-dimensional vascularized tissue engineering bone.

4. The method for preparing a three-dimensional vascularized tissue-engineered bone according to claim 3, wherein the lower 1/2 part of the scaffold is a low concentration gradient hydrogel module; the upper 1/2 part of the bracket is a high concentration gradient hydrogel module.

5. The method of claim 3, wherein the hydrogel pre-mixed solution is re-suspended with the mesenchymal stem cells and the vascular endothelial cells before UV curing.