Patent Application
20220331483
The present invention relates to a material for inducing nerve regeneration in a transplantation site, wherein the material comprises a three-dimensional cell structure having a thickness of at least 300 μm.
Previously, a poorly compliant bladder and a cystatrophia animal model have not been established in the fundamental research area of urology. On the other hand, a radiation-injured bladder model similar to such a poorly compliant bladder or cystatrophia has been established by applying radioactive rays to a rat bladder. In addition, regarding a freeze-injured bladder model, the problem still remains that the freeze-injured bladder model is reversible and thus, an injured site is recovered as a result of natural healing.
The poorly compliant bladder or cystatrophia is irreversible. Hence, when an irreversible damage is given to the bladder by irradiation, smooth muscle cells are reduced and also, neurons are significantly reduced from the tissues of the radiation-injured bladder model, as in the case of freezing injury (Non Patent Literature 1).
Non Patent Literature 1: Imamura, Ishizuka, Zhang, Hida, Gautam, Kato, Nishizawa. Bone marrow-derived cells implanted into radiation-injured urinary bladders reconstruct bladder tissues in rats. Tissue Engineering Part A, 18: 1698-1709, 2012
In the present invention, it has been desired to develop a regenerative medicine material, which is capable of recovering a reduction in neurons associated with a poorly compliant bladder or cystatrophia.
As a result of intensive studies directed towards achieving the aforementioned object, the present inventor has found that a three-dimensional cell structure having a thickness of at least 500 μm functions as a tissue regeneration-inducing material in the transplantation site, thereby completing the present invention.
Specifically, the present invention is as follows.
(1) A material for inducing nerve regeneration in a transplantation site, wherein the material comprises a cell structure having a thickness of at least 300 μm that is constructed by stacking the spheroids of bone marrow-derived cells, adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, on a needle-shaped body arranged on a substrate.
(2) The nerve regeneration-inducing material according to the above (1), wherein the transplantation site is a tissue or an organ comprising a smooth muscle layer.
(3) The nerve regeneration-inducing material according to the above (2), wherein the tissue or organ comprising a smooth muscle layer is at least one selected from the group consisting of bladder, ureter, urethra, penis, uterus, vagina, spermatic duct, and fallopian tube.
(4) The nerve regeneration-inducing material according to any one of the above (1) to (3), which further provides at least one selected from the group consisting of regeneration of microvessels, normalization of collagen fibers, the improvement of hypoxia, and the improvement of basal pressure, threshold pressure, micturition pressure, voiding interval, micturition volume, and residual volume in the bladder.
(5) A material for recovering nerve function in a transplantation site, wherein the material comprises a cell structure having a thickness of at least 300 μm that is constructed by stacking the spheroids of bone marrow-derived cells, adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, on a needle-shaped body arranged on a substrate.
(6) The function-recovering material according to the above (5), wherein the transplantation site is a tissue or an organ comprising a smooth muscle layer.
(7) The function-recovering material according to the above (6), wherein the tissue or organ comprising a smooth muscle layer is at least one selected from the group consisting of bladder, ureter, urethra, penis, uterus, vagina, spermatic duct, and fallopian tube.
(8) The function-recovering material according to the above (6) or (7), which further provides at least one selected from the group consisting of regeneration of microvessels, normalization of collagen fibers, the improvement of hypoxia, and the improvement of basal pressure, threshold pressure, micturition pressure, voiding interval, micturition volume, and residual volume in the bladder.
According to the present invention, a material for inducing nerve regeneration in a transplantation site is provided. When the nerve regeneration-inducing material of the present invention is transplanted into a target tissue or organ, nerve regeneration that provides the recovery of the function of the tissue or organ can be induced in the transplantation site.
The present invention relates to a material for promoting regeneration of the nerve in a tissue or an organ of interest by transplantation of a bone marrow-derived cell structure into the tissue or organ of interest. In the present invention, it is also possible to use adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, instead of the bone marrow-derived cells. The present invention relates to a material for recovering nerve tissues or a material for improving the function of nerve tissues, wherein the material comprises a cell structure having a thickness of at least 300 μm that is constructed by stacking the spheroids of bone marrow-derived cells, adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, on a needle-shaped body arranged on a substrate.
The present inventor has attempted to transplant a bone marrow-derived cell structure into an injured bladder, so as to regenerate functional bladder tissues.
The cells used to produce the present cell structure are, for example, bone marrow-derived cells. Bone marrow-derived cells means cells obtained by subjecting cells collected from the bone marrow to a primary culture in a collagen-coated culture plate, then adhering and extending the cells in the culture plate, and then allowing the cells to proliferate. Such marrow-derived cells may be either a mixture of multiple types of cells that mainily include mesenchymal cells comprising stem cells, or cells separated by a cell sorter using multiple cell markers, etc. In the present invention, it is also possible to use adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, instead of the bone marrow-derived cells.
The above-described bone marrow-derived cells, adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells are cultured or maintained in a medium suitable for individual types of cells, and the cells are prepared at time of use. Besides, various types of antibiotics, fetal bovine serum, and the like can be added to the medium, as necessary.
Thus, when the bone marrow-derived cells are continuously cultured, the cells are aggregated to form a cell aggregate, namely, a spheroid. The ability of the cells to form a spheroid can be examined, for example, by a morphological examination using an optical microscope.
A method of arranging the cells in any given three-dimensional space to produce a three-dimensional structure of the cells has been known (WO2008/123614). This method comprises arranging a needle-shaped body in the shape of a needle point holder on a substrate, and then sticking a cell mass into the needle-shaped body.
In the present invention, by utilizing the above-described method, spheroids are stacked to produce a three-dimensional cell structure (three-dimensional structure). Since an automatic stacking robot has already been known to realize the above-described method (Bio 3D Printer “Regenova” (registered trademark), CYFUSE BIOMEDICAL K.K.), the three-dimensional structure is preferably produced using this robot.
The number of spheroids arranged and the shape of spheroids arranged are not particularly limited, and these are arbitrarily determined.
In addition, the thickness of a cell structure produced is set to be, at least, 300 μm. The thickness of the obtained structure is, for example, 300 μm to 1800 μm, 500 μm to 1500 μm, 600 μm to 1200 μm, or 600 μm to 1800 μm. By setting the thickness of the cell structure within the aforementioned range, not only paracrine effects are obtained upon the transplantation of the bone marrow cells, but also, blood vessels are induced to the transplanted tissues so as to reduce a fibrotic lesion, and further, the transplanted bone marrow cells are directly differentiated into tissues that constitute bladder tissues. It is expected that the improvement of a fibrotic lesion and reconstruction of bladder tissues will progress integrally. With regard to adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, and umbilical cord blood-derived cells, the number of cells arranged and the shape of the cells arranged can be set to be the same as those for the marrow-derived cells.
The three-dimensional cell structure that is formed from bone marrow-derived cells, adipose tissue-derived cells, dental pulp-derived cells, amnion-derived cells, placenta-derived cells, umbilical cord-derived cells, or umbilical cord blood-derived cells, as described above, is also referred to as “the cell structure of the present invention.”
Next, the cell structure of the present invention is transplanted into the tissue or organ of a recipient patient (subject animal).
The transplantation site is not particularly limited, as long as it is a tissue or an organ, in which regeneration of the nerve is desired. In addition, the transplantation method is not particularly limited, and any given method is applied herein.
The tissue or organ as a target of the transplantation of the cell structure of the present invention is a tissue or an organi comprising a smooth muscle layer. Examples of such a tissue or an organ may include bladder, ureter (upper urinary tract), urethra (lower urinary tract), penis, uterus, vagina, spermatic duct, and fallopian tube.
As described above, when the cell structure of the present invention is transplanted into a transplantation site of interest, a smooth muscle layer is reconstructred in the transplantation site, and at the same time, the nerve is regenerated.
After completion of the transplantation, whether or not a predetermined nerve is regenerated is confirmed. In the case of a human, for example, this confirmation is carried out by performing a non-invasive urodynamic test, so that the recovery of bladder functions associated with nerve regeneration can be clarified. When peripheral nerves that govern the functions of the bladder (wherein the sympathetic nerve is a hypogastric nerve, the parasympathetic nerve is a pelvic nerve, and the somatic nerve is a pudendal nerve) are regenerated, appropriate collection of urine and micturition can be carried out. Specifically, the bladder is extended (relaxed), the urethra is contracted, and the amount of urine collected is increased at the time of first desire to urinate (i.e., a desire to urinate for feeling that a certain amount of urine is accumulated in the bladder, and that urine can be retained for a while), and involuntary contraction of the detrusor muscle (i.e., bladder contraction that is regardless of one's intention) is disappeared, so that sufficient collection of urine (250 ml or more) can be carried out and thus, micturition can be voluntarily carried out. Moreover, upon micturition, the bladder is contracted, and the urethra is relaxed, so that the collected urine can be completely urinated without interruption of the micturition. That is to say, nerve regeneration can be evaluated by confirming whether or not the bladder and the urethra cooperatively perform conflicting movements and as a result, appropriate urine collection and micturition can be carried out.
The cell structure of the present invention is transplanted into a bladder that has been injured by irradiation, and infiltration of vascular endothelial cells is thereby caused to the recipient, so that microvessels can be regenerated.
When the cell structure of the present invention is transplanted into tissues that are under hypoxic conditions, the cells are engrafted, so that the hypoxic conditions of the recipient tissues can be improved. Accordingly, the cell structure of the present invention can be applied to a pathologic condition, in which a fiber layer is formed on histological tissues as a result of proliferation and/or accumulation of fibroblasts or fibroblast-like tissues and the cells constituting the tissues are under hypoxic conditions.
When the cell structure of the present invention is transplanted to a disease exhibiting such findings that a collagen layer, in which collagen-generating cells are detected by histopathology or a non-invasive in vivo imaging technique, is broken, swollen, enlarged or scattered, the cells are engrafted, so that the collagen fibers of the recipient tissues can be normalized.
When the cell structure of the present invention is transplanted to a disease exhibiting such findings that a nerve, for example, a peripheral nerve disappears, the cells are engrafted, neurons are infiltrated into the recipient tissues, and the functions are thereby recovered. This recovery can be observed by histopathology or a non-invasive in vivo imaging technique.
Cells that express a PH4B protein serving as a fibrosis marker are detected according to a non-invasive in vivo imaging technique (for example, modified luminescence detection, etc.). This detection method can be broadly utilized as a diagnostic marker for the treatment of fibrosis, without being limited to urologic diseases.
Cells that express an HIF1α protein that is a marker that indicates the hypoxic conditions of living tissue are detected according to a non-invasive in vivo imaging technique (for example, modified luminescence detection, etc.). This detection method can be broadly utilized as a diagnostic marker that indicates the hypoxic conditions of tissues, without being limited to urologic diseases.
When the cell structure of the present invention is transplanted to a neuropathic disease having such findings as the disappearance of peripheral nerves, a disease having such findings that acetylcholinesterase-positive cells are reduced or are not detected, or a disease having such findings that CGRP-positive afferent neurons are reduced or are not detected, the cells are engrafted and the neurons are infiltrated into the recipient tissues, so that the disease can be recovered. As a result, biochemical measurement values, namely, basal pressure, threshold pressure, micturition pressure, voiding interval, micturition volume, and residual volume are improved.
Hereinafter, the present invention will be more specifically described in the following examples. However, these examples are not intended to limit the scope of the present invention.
Twenty two female 10-week-old Sprague-Dawley (SD) rats (Japan SLC Inc., Shizuoka, Japan) were used as recipients. As donors for bone marrow cells, six 17-week-old Tg-SD rats (Japan SLC Inc.), which had been transfected with a green fluorescent protein (GFP), were used. All of the rats were bred with voluntarily feedable food and water under a 12-hour alternative light and dark cycle. After completion of each experiment, the rats were euthanized with an excessive amount of pentobarbital sodium solution (Kyoritsu Seiyaku Corporation, Tokyo, Japan). All of the animals were treated in accordance with the guidelines of National Institute of Animal Health and the guidelines approved by Animal Ethics Committee, School of Medicine, Shinshu University.
The bladder was injured with radiation rays as follows.
The recipient SD rats were anesthetized with a pentobarbital sodium solution (Kyoritsu Seiyaku Corporation) in an amount of 40 mg/kg of body weight. Thereafter, the body of each rat was protected with a shield made of iron, except for a circle with a diameter of 1 cm, having the pubis as a boundary. Thereafter, the pubis region including the bladder (exposure region) was irradiated with radiation doses of 2 Gy, once a week, continuously for 5 weeks. After completion of the final radiation exposure, the rats were bred for 2 weeks. Three days before transplantation, the irradiated rats were subjected to an immunosuppressive treatment using cyclosporine (Novartis International AG, Basle, Switzerland) in an amount of 15 mg/kg of body weight, and 6α-methylprednisolone (Sigma-Aldrich, St. Louis, Mo.) in an amount of 2 mg/kg of body weight. Two weeks after the final radiation treatment, the treated rats were used as recipient animals.
Bone marrow-derived cells were prepared as follows.
Both femurs were collected from Tg-SD rats used as donors, into each of which a GFP gene had been introduced, and were then suspended in 10 ml of a medium, Dulbecco's Modified Eagle Medium (DMEM) High Glucose (Gibco, Thermo Fisher Scientific KK, Kanagawa, Japan), supplemented with 15% fetal bovine serum (BioWest, Nouaille, France) and 0.1% penicillin-streptomycin solution (Gibco). The cells were seeded on a 10-cm culture plate (ASAHI TECHNO GLASS, Shizuoka, Japan) coated with type I collagen and were then cultured for 7 days. When the cells became confluent, the adhering and proliferating bone marrow-derived cells were transferred into a 225-cm2 culture flask (Asahi Techno Glass) coated with type I collagen, for subculture. In order to obtain a sufficient amount of cells, the subculture was carried out three times or four times.
After the bone marrow-derived cells had reached confluent as a result of the subculture performed three times or four times, the cells showed a relatively uniform spindle shape and were positive to STRO-1 as a mesenchymal cell marker. The bone marrow-derived cells were harvested, and were then suspended at a concentration of 4.0×105 cells/ml in a spheroid-forming medium consisting of DMEM Low Glucose (Gibco) supplemented with 10% standard fetal bovine serum (BioWest) and 1.0% penicillin-streptomycin solution. In order to form spheroids, the cell suspension (4.0×104 cells/0.1 ml) was seeded into each well of a 96-well U-shaped plate (Sumitomo Bakelite Co., Ltd., Tokyo, Japan), and were then cultured in a spheroid-forming medium at 37° C. in a 5% CO2 atmosphere for 2 to 4 days. As a result, each of the 96 wells formed a single spheroid.
Subsequently, using a 3D bioprinting robot system, Regenova (Cyfuse Biomedical KK, Tokyo, Japan), bone marrow-derived cell spheroids were stacked to produce a three-dimensional structure. Regenova collected a spheroid from each of the 96 wells, and thereafter, the thus collected spheroids were inserted into a 9×9 microneedle array (approximately 5×5 mm,
Two weeks after the final radiotherapy, the recipient rats were anesthetized by both a pentobarbital sodium solution and inhalation of 2% to 3% sevoflurane (Mylan Inc., Osaka, Japan). The irradiated bladder was exposed (
Two and four weeks after the transplantation of the bio-processed structure (2 weeks, n=6; 4 weeks, n=4) or the sham surgery (n=6, in both periods), a bladder measurement test was carried out.
Two days before an intravesical pressure test, a polyethylene catheter was inserted into the bladder. The bladder measurement test was carried out for approximately 30 minutes on non-anesthetized and non-restrained rats that were each placed in a metabolic cage. A normal saline at room temperature was injected into the bladder at a rate of 10 ml/h through the catheter. The bladder contract and the micturition volume were simultaneously recorded on a pen-writing oscillograph. The following intravesical pressure parameters were measured: basal pressure, threshold pressure, micturition pressure (cmH2O), voiding interval (minute), and micturition volume (ml). The residual volume (ml) was calculated by subtracting the micturition volume from the amount of the normal saline injected.
After completion of the bladder measurement test, the bladder was collected for histological and immunohistochemical examination (as described below). When significant adhesion of the adipose tissues was found, in order to avoid damage caused by an attempt to eliminate the adhering tissues, the bladder was collected together with the adipose tissues that adhered thereto.
The trimmed bladder was immobilized and was embedded in paraffin, and thereafter, the resultant was cut into continuous sections with a thickness of 5 μm. For the histological and immunohistochemical examination, the sections were stained with hematoxylin and eosin (HE), Masson's trichrome, enzyme-labeled acetylcholinesterase antibody (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), or Picrosirius Red.
For the immunohistochemical examination, the sections were stained with a GFP antibody (1:500, Mouse Monoclonal, Lifespan Biosciences, Inc., Seattle, Wash., USA). As a result, bone marrow-derived cells constituting a bio-processed structure were detected. The GFP antibody was detected by a secondary antibody consisting of Alexa fluor 488-bound donkey anti-mouse IgG (1:250, Molecular Probes, Eugene, Oreg., USA). Subsequently, the GFP antibody-stained sections were double-stained with an antibody against alpha-smooth muscle actin used as a smooth muscle cell marker (SMA, 1:100, Mouse Monoclonal, Progen Biotechnik GmbH, Heidelberg, Germany), or a calcitonin gene-related antibody. As a marker for afferent neurons, a CGRP peptide (1:500, Guinea Pig Monoclonal, Progen Biotechnik GmbH) was used.
These were detected by a secondary antibody consisting of donkey anti-mouse or anti-guinea pig IgG, conjugated with Alexa fluor 594 (1:250 in each case, Molecular Probes). Otherwise, other sections were stained with an SMA antibody, and was then detected by a secondary antibody consisting of Alexa fluor 594 (1:250, Molecular Probes)-conjugated donkey anti-mouse IgG.
The SMA antibody-stained section was double stained with collagen prolyl hydroxylase beta (P4HB, 1:50, Mouse Monoclonal, Novus Biological, Inc.) that was an enzyme essential for the synthesis of all collagens, and an antibody against hypoxia inducible factor 1α (HIF1α) that was a cell mediator in hypoxic response (1:50, Rabbit Polyclonal, Proteintech Group, Inc., Rosemont, Ill., USA).
The anti-P4HB antibody and the anti-HIF1α antibody were detected by a secondary antibody consisting of donkey anti-mouse or anti-rabbit IgG, conjugated with Alexa fluor 594 (1:250 in each case, Molecular Probes). The immunofluorescent section was stained, in comparison to nuclear staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 5 μg/ml, Molecular Probes).
The results were shown with a mean value±standard deviation. A statistical difference was determined using Excel (registered trademark) Statistical Program (ESUMI Co., Ltd., Tokyo, Japan). A comparison was made by non-repeated measure analysis of variance (ANOVA). A P value of less than 0.05 was determined to be statistically significant.
A cell structure that had not been used in transplantation was prepared, separately, and then, this cell structure was histologically examined. There were no disordered defects in the center of the structure (
Survival of Cell Structure Transplanted into Irradiated Bladder
Two weeks after the transplantation of a bio-processed structure and the control surgery of a false structure, the control bladder did not show at all the recovery of the incision made on the anterior wall of the urinary bladder (
A different smooth muscle layer was present in the bladder tissues near the transplanted structure (
Four weeks after the sham surgery, the surgical site was excised and confirmed. As a result, the surgical site was hardly recovered, compared with the surgical site 2 weeks after the surgery (
The present inventor has conducted immunohistochemical examination and histological examination, and has observed bladder tissues around the boundary between the recipient tissues and the transplanted cell structure. In addition, the present inventor has also observed bladder tissues around the excised region of the bladder subjected to a sham surgery.
Two weeks after the control surgery, acetylcholinesterase-positive cells (
In the sham surgery group, even 4 weeks after the surgery, acetylcholinesterase-positive cells were not clearly shown (
Besides, both 2 weeks and 4 weeks after the surgery, differentiation of the bone marrow-derived cells constituting the structure into CGRP-positive cells (afferent neurons) was not found in either the transplanted structure or the recipient bladder tissues. It was unknown whether the neurons were induced from the periphery as a result of the transplantation of the structure, or the cells that barely survived after irradiation were recovered and proliferated. Anyway, it was confirmed that the neurons of the recipient were regenerated (recovered) as a result of the transplantation of the structure, and thus that the bladder functions were improved.
Two weeks after the sham surgery, collagen fibers were infiltrated into the spaces of extracellular matrixes comprising injured smooth muscle layers (
Four weeks after the sham surgery, collagen fibers are significantly enlarged, and occupied the extracellular space in the absence of a smooth muscle layer (
In contrast, collagen fibers in the recipient bladder tissues 4 weeks after the transplantation of the cell structure were integrated among the clusters of smooth muscle cells (
In the intravesical pressure test conducted 2 weeks after the sham surgery or the transplantation of the cell structure, the micturition patterns were similar to each other. Four weeks after the sham surgery, the voiding interval of the control rats was less than 5 minutes (irregular voiding interval), and the micturition volume thereof was less than 1 ml, which showed significant symptoms of frequent micturition (
Individual micturition parameters were assumed from the intravesical pressure chart. Two weeks after the sham surgery or the transplantation surgery of the cell structure, basal pressure, threshold pressure, micturition pressure, voiding interval, and micturition volume were measured. Between the sham surgery control group and the cell structure transplantation group, there was no significant difference (
Four weeks after the sham surgery, the basal pressure (
However, regarding the voiding interval (
In terms of the basal pressure, the threshold pressure, and the micturition pressure 4 weeks after the sham surgery group or the transplantation of the cell structure, there was no significant difference between the sham surgery group and the cell structure transplantation group (
These micturition parameters in the cell structure-transplanted bladder were almost the same as those in the normal bladder that had not been irradiated with radioactive rays. These results demonstrated that the cell structure-transplanted rats are partially prevented from reductions in both urine collection function and micturition efficiency that occur in radiation-injured bladders.
The present inventor has studied a method of assisting the structural and functional recovery of the lower urinary tract that is mainly composed of the bladder and the urethra. Then, the present inventor has applied a tissue engineering methodology of utilizing a combination of biochemical factors based on cells, biological materials, and microenvironments in the target tissues and organs of a recipient. At current, biofabrication realized by a 3D bioprinter has been reported as a new biotechnology. Accordingly, we have incorporated this new biotechnology into our tissue engineering methodology. This methodology is referred to as “next-generation tissue engineering.”
By using the 3D bioprinting robot system, Regenova, a self-assembled tissue-like structure consisting of bone marrow-derived cells was biofabricated. The cell structure of the present invention has several advantages, which are not found in either a cell injection method or a cell sheet method. Firstly, the present cell structure has a thickness and a strength that are sufficient for facilitating the handling for transplantation. Secondly, the present cell structure can be directly transplanted into recipient tissues. Thirdly, the 3D conformation imitates naturally occurring tissues, and provides an intracellular contract for promoting self-assembly. Finally, the present cell structure has biocompatibility that is higher than that of an artificial material.
Regarding the effect of the cell structure on recipient tissues, a paracrine effect on the microenvironment of the recipient tissues adjacent to the cell structure is expected. Accordingly, a histological change at the boundary surface between the structure to be transplanted and the recipient tissues is important. The most important result is that the transplanted structure survived in the recipient tissues, and that blood vessels grew and extended from the adjacent recipient tissues into the cell structure.
While surrounding the extended blood vessels, the bone marrow-derived cells in the cell structure were differentiated into smooth muscle cells. Four weeks after the transplantation, those smooth muscle cells form a cluster thereof at the outer edge of the extended blood vessels surrounding them and the transplanted structure. In the transplanted cell structure, HIF1α-positive cells exhibiting hypoxia were sparsely distributed. Hence, the microenvironment surrounding the blood vessels extended from the recipient tissues supports differentiation of smooth muscle cells from the bone marrow-derived cells and cluster formation of the smooth muscle cells, and it shows that the smooth muscle cells have been differentiated from the bone marrow-derived cells.
It has been reported that a signal pathway including the expression of hypoxia-dependent HIF1α causes fibrosis in an injured bladder (Ekman, M. et al., Lab Invest 94, 557, 2014, Iguchi, N. et al., Am J Physiol Renal Physiol, 313, F1149, 2017, Wiafe, B. et al., In Vitro Cell Dev Biol Anim 53, 58, 2017.). However, at the time points that were 2 weeks and 4 weeks after the surgery, the number of HIF1α-positive cells as hypoxia markers was small in the recipient tissues of a cell structure-transplanted bladder, in comparison to that in a false structure control. In addition, significant fibrosis was not developed in the structure-transplanted bladder, or the structure-transplanted bladder comprised a large number of P4HB-positive cells.
The presence of the cell structure and the relevant growth of blood vessels from the surrounding tissues into the cell structure create an optimal microenvironment, and reduce or eliminate hypoxia that relates to wound. Consequently, the HIF1α pathway is activated only transiently and/or to the minimum. Accordingly, it is considered that decomposition of extracellular matrix collagen mediated by the P4HB pathway is limited. This explains that fibrosis is significantly reduced (improved) in a bladder into which the cell structure has been transplanted. Moreover, the histological findings of such a cell structure-transplanted bladder were similar to the histological findings of a normal bladder that has not been irradiated with radioactive rays.
Furthermore, as shown in the previous studies, it was shown that the transplantation of the cell structure into the irradiated bladder induces the recovery of the bladder functions (Imamura, T. et al., Tissue Eng Part A 18, 1698, 2012, Imamura, T. et al., Tissue Eng Part A 21, 1600, 2015.). Four weeks after the transplantation, the cell structure-transplanted rats did not exhibit significant frequent micturition symptoms. The voiding interval and the micturition volume were higher in the cell structure-transplanted rats than those in control rats. Moreover, both 2 weeks and 4 weeks after the transplantation, the residual volume in the cell structure-transplanted rats was smaller than the residual volume in the control rats. The transplantation of the cell structure improved both urine collection function and micturition efficiency, and recovered them to the levels of a normal bladder. Furthermore, these results suggest that the transplanted structure may suppress progressive irradiation damage. It is considered that the improvement or suppression of progressive irradiation damage is associated with alleviation of radiation-induced frequent micturition symptoms.
Differing from the advantages of the present cell structure that exceeds a single-type cell direct injection method and a cell sheet patch transplantation method, the cell structure enables the delivery of a larger number of cells to the target site.
With regard to the construction of the cell structure, 243 spheroids were inserted into an area from the middle part to the upper part of a 9×9 microneedle array. Each spheroid was formed with 4×104 cells. As a result, the number of cells in each cell structure was approximately 1×107 cells (243 spheroids×4×104 cells/spheroid=approximately 1×107 cells). This number of cells is approximately 100 times greater than the number of cells used in either the cell injection method or the cell sheet method.
Even two weeks after the transplantation, which was a half of the period in the previous studies conducted by the present inventor, the bladder functions of the cell structure-transplanted rats, except for the residual volume, were not changed from those of the sham surgery control rats. Accordingly, strictly speaking, the recovery 2 weeks after the transplantation does not seem to be associated with the number of the transplanted bone marrow-derived cells. The data provided by the present inventors suggest that, regarding reconstruction of functional tissues, a certain recovery period, namely, at least 4 weeks, should be required for cell replication, differentiation, and tissue construction, in the case of the experimental models used herein. During such a recovery period, the transplanted cell structure is able to replace and substitute for damaged tissues, so that the present cell structure could provide regeneration effects, which had not been elucidated by the direct injection method and the cell sheet method.
In conclusion, for the biofabrication of a cell structure consisting of bone marrow-derived cells, the present inventor has used a 3D bioprinting robot system. The cell structure survived after it had been transplanted into the irradiated rat bladder, and blood vessels were infiltrated from the recipient tissues adjacent to the cell structure into the cell structure. The bone marrow-derived cells constituting the cell structure were differentiated into smooth muscle cells, which then formed a cluster thereof. Although the transplanted cells were not differentiated into neurons, the regenerated neurons were present in the recipient bladder tissues. In the bladder tissues into which the cell structure had been transplanted, significant fibrosis associated with HIF1α-positive cells and P4HB-positive cells was not developed. Four weeks after the transplantation of the cell structure, the frequent micturition symptoms of the rats were improved, and the residual volume was reduced. Therefore, the present cell structure will become a great tool for treating patients having severe lower urinary tract symptoms that are caused by the damage of the bladder.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-093184 | May 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/019234 | 5/14/2020 | WO |
