In vivo study of the angiogenesis potential of bone marrow-derived mesenchymal stem cell aggregates in their niche like environment

Abstract

Background:

Sufficient blood vessel formation in bioengineered tissues is essential in order to keep the viability of the organs. Impaired development of blood vasculatures results in failure of the implanted tissue. The cellular source which is seeded in the scaffold is one of the crucial factors involved in tissue engineering methods.

Materials and methods:

Considering the notable competence of Bone Marrow derived Mesenchymal Stem Cell aggregates for tissue engineering purposes, in this study BM-aggregates and expanded BM-MSCs were applied without any inductive agent or co-cultured cells, in order to investigate their own angiogenesis potency in vivo. BM-aggregates and BM-MSC were seeded in Poly-L Lactic acid (PLLA) scaffold and implanted in the peritoneal cavity of mice.

Result:

Immunohistochemistry results indicated that there was a significant difference (p < 0.050) in CD31+ cells between PLLA scaffolds contained cultured BM-MSC; PLLA scaffolds contained BM-aggregates and empty PLLA. According to morphological evidence, obvious connections with recipient vasculature and acceptable integration with surroundings were established in MSC and aggregate-seeded scaffolds.

Conclusion:

Our findings revealed cultured BM-MSC and BM-aggregates, capacity in order to develop numerous connections between PLLA scaffold and recipient’s vasculature which is crucial to the survival of tissues, and considerable tendency to develop constructs containing CD31+ endothelial cells which can contribute in vessel’s tube formation.

Keywords

Angiogenesis bone marrow-derived cell aggregates cultured bone marrow-derived mesenchymal stem cells Poly-L Lactic Acid scaffold

Introduction

Ageing, disease and trauma as the major causes of tissue damage and injury impose enormous operational and financial burden on the health care system and individuals.¹ Researchers in two main areas of regenerative medicine; cell therapy, and tissue engineering are exploring to find efficient replacements and treatments for damaged tissues.² Despite all efforts, the clinical achievements have been limited due to some obstacles. One of the most important problems is the insufficient angiogenesis which prevents the efficient integration and viability of implanted tissue. Features of cell source, characteristics of the scaffold, and the interaction of cell source and the scaffold are the most critical aspects of tissue engineering.^3–5 Mesenchymal Stem Cells (MSCs) are one of the most prevalent types of adult stem cells which are used in several studies.^6–8 Furthermore several completed clinical trials are using MSCs for the treatment of bone and tissues disease, for example knee osteoarthritis, dental implants, maxillary bone cysts, etc.^9–11 Based on our previous studies MSCs are not isolated in a niche; in fact, they are in the form of cell aggregates. We have extracted MSC’s preserved in their native niche-like environment assumed as the stromal component of haematopoietic stem cell niches.^12,13 In combination with 3-D scaffold subsequent to transplanting into the mice, native BM structure was observed in transplanted bioengineered tissue.¹⁴ In addition to the remarkable competence of BM-aggregates they are available in urgent cases and no expansion or passage is needed, in contrary with expanded MSCs; furthermore, in the form of cell aggregates, haematopoietic and mesenchymal stem cells reside in their original and their behaviour remains much the same as their in vivo behaviour. Mentioned characteristics of BM-aggregates make them appealing for tissue regeneration purposes so we assessed osteogenic differentiation potential and feasibility of their attachment to scaffold which revealed BM-aggregate’s faster osteogenic differentiation than expanded MSCs.¹⁵ Promising properties of BM-aggregate considering the necessity of angiogenesis in bone tissue engineering and other transplanted tissues led us to investigate the angiogenesis potential of BM-aggregates. This research was conducted to evaluate the angiogenesis potential of BM-aggregate versus cultured BM-MSC and their interaction in contact with Poly L-Lactic Acid (PLLA) scaffold comparing with an empty scaffold. PLLA is a degradable, accessible, biocompatible scaffold with favourable mechanical attributes.^16,17 In vivo researches would be essential to explore the practical findings to promote angiogenesis in transplanted tissues.

Materials and methods

Fabrication of Nanofiber PLLA scaffold: Electrospinning fabrication method was used to construct the scaffold, as previously described.^14,18 Briefly, a solution of PLLA (Sigma-Aldrich, St. Louis, MO, USA) in dichloromethane (Merck, Darmstadt Germany, 12% w/v) was prepared. A 20 KV voltage was applied as the force to throw the PLLA droplets to the collector surface which consequently were deposited as scaffold ultrafine fibres.

Assessment of the PLLA scaffold: The Gold coated empty scaffolds and seeded ones were evaluated by Scanning Electron Microscopy (SEM, Seron Technology, AIS2100, Amir Kabir University) to determine the morphology and cellular attachment capacity. 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT) was used to assess the biocompatibility of the scaffold and the determination of its effects on the proliferation of attached cells.¹⁴ BM-MSC seeded scaffolds were treated with (MTT) (Bio idea, Tehran, Iran) 1/10 diluted in serum-free cell culture (without phenol red). The yellow colour of MTT turns to purple in live cells and the amount of produced purple colour is directly related to cell counts. Subsequent 4-h incubation in 37° and 5% CO₂, 100 λ dimethyl sulfoxide (DMSO, Bio idea, Tehran, Iran) was added, and after 10 min the optical density was measured at 570 nm. All the aforementioned steps have been occurred in the 1st, 3rd, 5th and 7th days after seeding and repeated three times. Nucleus staining with 4′, 6-diamidino-2-phenylindole, (DAPI, Sigma-Aldrich, St. Louis, MO, USA) also was performed to determine if the scaffold is appropriate for the cells to proliferate and increase on the scaffold. 1 and 7 days after seeding the BM-MSC on the scaffold, they were fixed in paraformaldehyde 4% and then DAPI was added to cells which were permeabilized by triton x-100 (Sigma-Aldrich, MO, USA). Then they were visualized by UV microscopy. (TE2000- S; Nikon-Eclipse).¹⁹

Animals: 8–10-week-old female c57BL/6(Pasteur Institute, Tehran, Iran) were used as the recipient of implants and donors of bone marrow aggregates and MSC. There were seven mice in every group as the recipients of the implants. WE considered the National Institutes of Health Guide for the Care and Use of Laboratory Animals, in all animal care and experiments.

Murine BM-Aggregates Extraction: The mice were sacrificed by cervical dislocation. Femurs and tibia were separated and after removing the fat and muscle both sides of them were cut, put in a microtube and centrifuged 1 min and 400g as described formerly.¹³ Acquired pellet diluted in heparinized phosphate-buffered saline (PBS, Gibco-BRL, Grand Island, NY ) and filtered (a double-layered 40 μm nylon mesh; BD Bioscience, San Jose, CA, USA). The aggregates that remained on the surface of the filter were collected and washed with PBS.

BM-MSC Extraction, Isolation and Characterization: After incubating the cell aggregates for 3 days, the flasks were washed and Dulbecco’s Modified Eagle Medium (DMEM, Gibco-BRL) was replaced. MSCs were attached to the flask and the medium was replaced every 3 days. WE applied BM-MSCs which were characterized in our previous study.¹³

Implantation: The recipient mice were divided into three groups. The sterilized 1*1.5 PLLA scaffolds were seeded by just one kind of cell source, MSC or aggregates, and the empty scaffolds roll-folded and respectively implanted separately into the peritoneal cavity of each group’s recipients. After 18 days implants were harvested and cut into three parts to prepare the lateral and medial sections.¹⁴

Immunohistochemistry (IHC) and Hematoxylin and Eosin (H&E) staining: The implants were fixed, dehydrated and paraffin-embedded blocks were prepared. 3 mm thick serial sections were prepared using a microtome. H&E staining has occurred following the routine protocols and for immunohistochemistry anti CD31 antibody (Abcam, Cambridge, USA) which is the marker of endothelial cells was used to determine the vessels endothelial.^6,7,20–23 this test was performed as previously described.^19,24

Image Analysis: Stained sections visualized using light microscopy (Olympus, BX5), 40× magnification investigating CD31+ areas. Image-Pro Plus (version 10.0.7, Media Cybernetics) was used to determine the dyed percent area of images.²⁵ the ratio of the dyed area (CD31 positive area) to the total area of the image in percent (named per area) was calculated using the software.

Statistical Analysis: Every implanted construct was harvested from every group and cut into three sections (from both sides and the middle of the scaffold in order to access most parts of the construct) five slides were randomly selected and visualized and then five images of every section were selected randomly. Finally, 225 images of each group were analysed; images were coded to avoid inclination. Thirty images of each group randomly were chosen and the difference between groups was analysed by running the Kruskal–Wallis test using SPSS 22, (IBM Corporation, Armonk, NY). p-values of less than 0.05 were considered significant.

Results

Biocompatibility and Porosity Evaluation of the Scaffold: Viability, homing, growth, proliferation and efficient interaction of cells are due to the well-manufactured, porous and biocompatible scaffold structure. SEM method was applied to determine the porous structure and uniform fibres with an average diameter of 994 ± .4 nanometer (Figure 1(a)). Biocompatibility assessed by scanning the surface of cell-contained scaffolds, demonstrated strongly attached cells spread among the fibres (Figure 1(b)). MTT assay quantification showed no significant difference in proliferation rate between cells expanded in the scaffold versus normal plate cultures, applying an independent T-test (p < 0.05) (Figure 1(c)). These results were confirmed with DAPI staining. Increased number of the fluorescent nucleus was distinguished comparing 7-day incubated cell-seeded scaffolds and plate cultivated ones with their first day of incubation (Figure 1(d)–(g)).

Figure 1.

Scaffold characterization. (a) SEM revealed the scaffold’s porous structure, fibre’s diameter was estimated 994 ± 0.4 nm (scale bar: 50 μm). (b) MSC strongly attached and spread among the fibres (scale bar: 100 μm). (c) MTT illustrated the proliferation rate of cells on 1, 3, 5 and 7 days. No significant difference observed between PLLA attached cells and tissue cultured plate (TCP), applying an independent T-test (p < 0.05). Error bars represent the Standard Deviation of mean. (d, e) MSC cultivated on scaffold and plate, treated by DAPI after 24 h, (f, g) and 7 days demonstrated an increased number of nucleus in both groups (scale bars: 50 μm).

Morphological Description of Harvested Transplanted Scaffolds: In this study, mouse bone marrow cell aggregates were extracted and seeded on PLLA scaffold as was done for in vitro expanded MSC, then the scaffolds implanted into the peritoneal cavity of recipient mice, in order to evaluate the angiogenesis potential of these two cell sources and their interaction in contact with PLLA scaffold in comparison with the empty scaffold. After 18 days, recipients were sacrificed and the scaffolds were removed. Empty PLLA was found to be surrounded by fascia and poorly connected to other abdominal organs (Figure 2(a) and (b)). Due to the biocompatible nature of the scaffold, moderate acceptance of even empty scaffold was expected. Aggregate contained scaffolds were connected to the organs and some vessels surrounded the structure from different sides especially the penetration of some vessels from holes in sides of the rolled scaffold was observed (Figure 2(c) and (d)) vessels were integrated into the scaffold (Figure 2(e)). MSC-seeded scaffolds were in good interaction with omentum vasculature and organs (Figure 2(f) and (g)) and many vessels penetrated and integrated into them (Figure 2(h) and (i)).

Figure 2.

Morphological description of harvested transplanted scaffolds after 18 days. (a, b) Empty PLLA was poorly connected to other abdominal organs. (c, d) Aggregate-seeded scaffolds were connected to the organs and some vessels surrounded the structure from different sides especially the penetration of some vessels from holes in sides of the rolled scaffold was observed, (e) these vessels were attached to the scaffold and penetrated the construct. (f, g) MSC-seeded scaffolds were in good interaction with omentum vasculature and organs (h, i) many vessels penetrated and integrated in to them.

IHC, H&E, Image Analysis, and Statistical Results: CD31 is a transmembrane protein expressed on endothelial cells such as vessels endothelium.²¹ IHC was performed to evaluate CD31+ cell amounts in each group. CD31+ regions were recognizable in all groups (Figure 3(a) and (b)) but CD31- regions were significant in empty PLLA group (the scaffolds which transplanted empty with no seeded cells) that (Figure 3(c)) was contrary for two other groups; especially this discrepancy was significant about the MSC group which showed intense CD31+ overall results. The structure of some vessels was distinguishable in MSC and aggregate-seeded scaffolds, the vessel’s CD31+ endothelial cells were apparent. The sections were visualized as before described and all efforts have been made to take images with the same quality and brightness. We tried to select the images with distinguishable scaffold fibres. Thirty images of each group were randomly selected and were analysed by applying image pro plus software to assess the ratio of dyed area to the total area of the image in percent, which defined per area of CD31+ regions. In order to compare the three nonparametric groups, Kruskal - Wallis test was used. The significant difference was defined between mean intensity per area of MSC-PLLA and Empty PLLA, as well as Aggregate-PLLA and Empty PLLA. The MSC difference was more significant (Figure 3(d)). Considering the p-values less than 0.05 were appointed significant. H&E staining demonstrated cell population spread within the scaffold fibres. Empty scaffold contained lower cell density in comparison with two other groups (Figure 3(e)–(g)).

Figure 3.

IHC, H&E, image analysis and statistical process. (a, b) More CD31+ cells (marked in red) comparing with empty scaffold (the scaffolds which transplanted empty with no seeded cells) were detected in MSC and aggregate seeded scaffolds. The structure of vessels and their CD31+ endothelial cells were apparent. (c) CD31+ areas of empty scaffolds (marked in red) were slight; (all scale bars: 50 μm). (d) The ratio of dyed area to the total area of image in percent, were analysed. The difference between per area means of MSC and aggregate-PLLA with empty PLLA were significant. p-Values less than 0.05 were appointed significant. Error bars represent Standard Deviation of mean. H&E staining demonstrated cell population spread within the scaffold fibres in (e) BM-aggregate seeded scaffolds (f) MSC seeded ones and (g) the empty scaffold which contained lower cell density in comparison with two other groups.

Discussion

According to morphological evidence, MSC and aggregate-seeded scaffolds contain abundant cellularity. Obvious connections with recipient vasculature and acceptable integration with surroundings were established that were not the same with empty PLLA. This apparent morphological difference besides the significant difference in CD31+ cells would indicate the angiogenesis potency of these cell sources and their efficient features to keep the viability of the implanted tissue. Our previous study agrees with these results, BM haematopoietic stem and progenitor cells niche-like units seeded in scaffold which existed 1 month in recipient showed native BM structure and efficient connection to the mouse vascular system was established while empty scaffolds were not connected to the recipient tissues and vasculature.¹⁴ The existence of cell populations in our empty scaffolds insists on the acceptable features of the scaffold and might be relevant to the appointed duration that scaffolds existed in recipients. Considering the undeniable necessity of angiogenesis and effectual connection between implanted tissue and recipient’s vasculature in order to keep the viability of the implant and cell population it contains; it would be expected that empty PLLA with poor connections become ablated during longest periods as well as the cited study’s evidence. More studies with longer durations are recommended.

MSCs have been applied in many angiogenesis investigations and several aspects of this kind of cells have been evaluated; researchers have co-cultured MSCs with different kinds of cells, for example, endothelial outgrowth cells or human umbilical vein endothelial cells, in order to analyse their effect on network formation or vasculogenesis ability of those cells^26–28; also they have been induced to differentiate to endothelial cells which are able to establish vessels.²² MSCs have been used with special inductive agents or scaffolds in vitro and in vivo in order to promote vasculogenesis in many tissues. In this study we applied expanded BM-MSC and BM-aggregates without any inductive agent or co-cultured cells, to investigate their own angiogenesis potency in vivo. In a study Matrigel-embedded cultured-MSCs were implanted subcutaneously into mice; elicited a robust angiogenic reaction, leading to a remarkable increase in vessel density. Researchers stated the in vivo vascular differentiation of MSCs might be related to the interaction between the cells and matrigel. It should be considered that matrigel contained a wide variety of elements, any of which might provoke signals promoting endothelial differentiation.¹⁷ In our study, we applied PLLA to make suitable matrices for cells and we hadn’t utilized differentiation-inductive agents.

Both aggregate-seeded and MSC-loaded scaffolds demonstrated angiogenesis tendency in order to establish numerous connections between PLLA scaffold and recipient’s vasculature, and considerable propensity to develop constructs containing CD31+ endothelial cells which can contribute to vessel’s tube formation. MSCs are multipotent adult stem cells that were used in tremendous researches and nominated as one of the most practical cell sources in many studies of regenerative medicine.^29,30 MSC Cultivation and in vitro expansion are essential in order to achieve a sufficient amount of cells which is needed in most clinical and research protocols,^12,31 while BM-aggregate is available in urgent cases with no need for expansion and passage which keep it acquitted from controversial subsequent of several expansions.^32,33 Also its interaction with the microenvironment and other niche components may prevent unfavourable changes.^12,13 Although the existence of several distinguishable vessel structures with CD31+ endothelial cells in aggregate contained scaffolds as well as the MSC ones, CD31+ percent area of the expanded MSC was higher. The amount of BM-aggregate which was seeded in the scaffold might modify this score and this issue should be involved, so we recommend applying BM-aggregates in different types of bioengineered scaffold furthermore optimizing the required concentration for further researches. Due to the notable osteogenic properties of BM-aggregates, we have introduced them as a suitable source for bone tissue engineering,¹⁵ according to angiogenesis potency which they have shown in this study and for the aforementioned reasons, BM-aggregate would be introduced as an applicable and noteworthy cell source in bone tissue engineering and other fields of regenerative medicine. It seems more studies are needed to investigate various characteristics of BM-aggregate.

Footnotes

Declaration of conflicting

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Isfahan University of Medical Sciences, Isfahan, Iran (grant number: 394768).

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