Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

The effect of decellularized cartilage matrix scaffolds combined with endometrial stem cell-derived osteocytes on osteochondral tissue engineering in rats.

Since decellularized tissues may offer the instructive niche for cell differentiation and function, their use as cell culture scaffolds is a promising approach for regenerative medicine. To repair osteochondral tissues, developing a scaffold with biomimetic structural, compositional, and functional characteristics is vital. As a result of their heterogeneous structure, decellularized articular cartilage matrix from allogeneic and xenogeneic sources are considered appropriate scaffolds for cartilage regeneration. We developed a scaffold for osteochondral tissue engineering by decellularizing sheep knee cartilage using a chemical technique. DNA content measurements and histological examinations revealed that this protocol completely removed cells from decellularized cartilage. Furthermore, SEM, MTS assay, and H&E staining revealed that human endometrial stem cells could readily adhere to the decellularized cartilage, and the scaffold was biocompatible for their proliferation. Besides, we discovered that decellularized scaffolds could promote EnSC osteogenic differentiation by increasing bone-specific gene expression. Further, it was found that decellularized scaffolds were inductive for chondrogenic differentiation of stem cells, evidenced by an up-regulation in the expression of the cartilage-specific gene. Also, in vivo study showed the high affinity of acellularized scaffolds for cell adhesion and proliferation led to an improved regeneration of articular lesions in rats after 4 weeks. Finally, a perfect scaffold with high fidelity is provided by the developed decellularized cartilage scaffold for the functional reconstruction of osteochondral tissues; these types of scaffolds are helpful in studying how the tissue microenvironment supports osteocytes and chondrocytes differentiation, growth, and function to have a good osteochondral repair effect.

Production and evaluation of decellularized extracellular matrix hydrogel for cartilage regeneration derived from knee cartilage.

Cartilage tissue engineering is the interdisciplinary science that will help to improve cartilage afflictions, such as arthrosis, arthritis, or following joints traumatic injuries. In the present work, we developed an injectable hydrogel which derived from decellularized extracellular matrix of sheep cartilage. Successful decellularization was evaluated by measuring the DNA, glycosaminoglycans (GAG), collagen contents, and histological analyses. There was a minor difference in GAG and collagen contents among natural cartilage and decellularized tissue as well as ultimate hydrogel. Rheological analysis showed that the temperature and gelation time of prepared hydrogel were 37°C and between 5 and 7 min, respectively. Mechanical properties evaluation indicated a storage modulus of 20 kPa. The results show that prepared hydrogel possessed cell-friendly microenvironment as confirmed via calcein staining and MTT assay. Also, cells were able to proliferate which observed by H&E and alcian blue staining. Cell attachment and proliferation at the surface of the decellularized hydrogel was apparent by Scanning Electron Microscope (SEM) images and microphotographs. Furthermore, the cells embedded within the hydrogel were able to differentiate into chondrocyte with limited evidence of hypertrophy and osteogenesis in utilized cells which proved by SOX9, CoL2, ACAN, and also CoL1 and CoL10 gene expression levels. In summary, the results suggest that developed novel injectable hydrogel from decellularized cartilage could be utilized as a promising substrate for cartilage tissue engineering applications.

Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: proliferation and differentiation to bone or cartilage.

Tissue engineering aims to take advantage of the ability of undifferentiated stem cells to differentiate into multiple cell types to repair damaged tissue. Photobiomodulation uses either lasers or light-emitting diodes to promote stem cell proliferation and differentiation. The present study aimed to investigate single and dual combinations of laser wavelengths on mesenchymal stem cells (MSCs). MSCs were derived from rabbit iliac bone marrow. One control and eight laser irradiated groups were designated as Infrared (IR, 810 nm), Red (R, 660 nm), Green (G, 532 nm), Blue (B, 485 nm), IR-R, IR-B, R-G, and B-G. Irradiation was repeated daily for 21 days and cell proliferation, osseous, or cartilaginous differentiation was then measured. RT-PCR biomarkers were SOX9, aggrecan, COL 2, and COL 10 expression for cartilage and ALP, COL 1, and osteocalcin expression for bone. Cellular proliferation was increased in all irradiated groups except G. All cartilage markers were significantly increased by IR and IR-B except COL 10 which was suppressed by IR-B combination. ALP expression was highest in R and IR groups during osseous differentiation. ALP was decreased by combinations of IR with B and with R, and also by G alone. R and B-G groups showed stimulated COL 1 expression; however, COL 1 was suppressed in IR-B, IR-R, and G groups. IR significantly increased osteocalcin expression, but in B, B-G, and G groups it was reduced. Cartilage differentiation was stimulated by IR and IR-B laser irradiation. The effects of single or combined laser irradiation were not clear-cut on osseous differentiation. Stimulatory effects on osteogenesis were seen for R and IR lasers, while G laser had inhibitory effects.

Correction to: Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: proliferation and differentiation to bone or cartilage.

In the originally published article, the name of the 3rd and 4th authors were labeled incorrectly. The correct names are Mohammadreza Baghaban Eslaminejad and Leila Taghiyar. Also, affiliation 4 has been corrected.

Injectable natural polymer compound for tissue engineering of intervertebral disc: In vitro study.

Intervertebral disc degeneration is recognized to be the leading cause for chronic low-back pain. Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation specially for intervertebral (IVD) affecting rate of regeneration success, in this study we assessed viscoelastic properties of a Chitosan-ß glycerophosphate-hyaluronic acid, Chondroitin-6-sulfate, type 2 of Collagen, gelatin, fibroin silk (Ch-ß-GP-HA-CS-Col-Ge-FS) hydrogel which was named as NP hydrogel that is natural extracellular matrix of IVD. Chitosan-based hydrogel was made in the ratio of 1.5%: 7%: 1%:1%:1%-1.5%-1% (Ch: ß-GP: HA-CS-Col-Ge-FS). Gelation time and other rheological properties were studied using amplitude sweep and frequency sweep tests. Also, the cytotoxicity of the hydrogel invitro assessed by MTT and trypan blue tests. Morphology of the hydrogel and attachment of NP cells were evaluated by SEM. Our result showed that NP hydrogel in 4°C is an injectable transparent solution. It started gelation in 37°C after about 30min. Gelation temperature of NP hydrogel was 37°C. Storage modulus (G') of this hydrogel at 37°C was almost constant over a wide range of strain. MTT and trypan blue tests showed hydrogel was cytocompatible. The obtained results suggest that this hydrogel would be a natural and cytocompatible choice as an injectable scaffold for using in vivo study of IVD regeneration.

Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

OBJECTIVES: The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). MATERIALS AND METHODS: In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. RESULTS: UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. CONCLUSIONS: PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy.

Bone marrow mesenchymal stem cell and vein conduit on sciatic nerve repair in rats.

BACKGROUND: Peripheral nerve repair with sufficient functional recovery is an important issue in reconstructive surgery. Stem cells have attracted extensive research interest in recent years. OBJECTIVES: The purpose of this study was to compare the vein conduit technique, with and without the addition of mesenchymal stem cells in gap-less nerve injury repair in rats. MATERIALS AND METHODS: In this study, 36 Wistar rats were randomly allocated to three groups: In the first group, nerve repair was performed with simple neurorrhaphy (control group), in the second group, nerve repair was done with vein conduit over site (vein conduit group) and in the third group, bone marrow stem cells were instilled into the vein conduit (stem cell group) after nerve repair with vein conduit over site. Six weeks after the intervention, the sciatic function index, electrophysiological study and histological examination were performed. RESULTS: All animals tolerated the surgical procedures and survived well. The sciatic function index and latency were significantly improved in the vein conduit (P = 0.04 and 0.03, respectively) and stem cell group (P = 0.02 and 0.03, respectively) compared with the control group. No significant difference was observed in sciatic function and latency between the vein conduit and stem-cell groups. Moreover, histological analysis showed no significant difference in regenerative density between these two groups. CONCLUSIONS: The results of this study showed that the meticulous microsurgical nerve repair, which was performed using the vein tubulization induced significantly better sciatic nerve regeneration. However, the addition of bone marrow mesenchymal stem cell to vein conduit failed to promote any significant changes in regeneration outcome.

Isolation and characterization of the progenitor cells from the blastema tissue formed at experimentally-created rabbit ear hole.

UNLABELLED: Objective(s) : Throughout evolution, mammalians have increasingly lost their ability to regenerate structures however rabbits are exceptional since they develop a blastema in their ear wound for regeneration purposes. Blastema consists of a group of undifferentiated cells capable of dividing and differentiating into the ear tissue. The objective of the present study is to isolate, culture expand, and characterize blastema progenitor cells in terms of their in vitro differentiation capacity. MATERIALS AND METHODS: Five New Zealand white male rabbits were used in the present study. Using a punching apparatus, a 4-mm hole was created in the animal ears. Following 4 days, the blastema ring which was created in the periphery of primary hole in the ears was removed and cultivated. The cells migrated from the blastema were expanded through 3 successive subcultures and characterized in terms of their potential differentiation, growth characteristics, and culture requirements. RESULTS: The primary cultures tended to be morphologically heterogeneous having spindly-shaped fibroblast-like cells as well as flattened cells. Fibroblast-like cells survived and dominated the cultures. These cells tended to have the osteogenic, chondrogenic, and adipogenic differentiation potentials. They were highly colonogenic and maximum proliferation was achieved when the cells were plated at density of 100 cells/cm2 in a medium which contained 10% fetal bovine serum (FBS). CONCLUSION: Taken together, blastema tissue-derived stem cells from rabbit ear are of mesenchymal stem cell-like population. Studies similar to this will assist scientist better understanding the nature of blastema tissue formed at rabbit ear to regenerate the wound.

Blastema from rabbit ear contains progenitor cells comparable to marrow derived mesenchymal stem cells.

Rabbits have the capacity to regenerate holes in their ears by forming a blastema, a tissue that is made up of a group of undifferentiated cells. The purpose of the present study was to isolate and characterize blastema progenitor cells and compare them with marrow mesenchymal stem cells (MSCs). Five New Zealand white male rabbits were used in the present study. A 2-mm hole was created in the animal ears. After 4 days, the blastema ring formed in the periphery of the hole was removed and cultivated. The cells were expanded through several subcultures and compared with the MSCs derived from the marrow of same animal in terms of in vitro differentiation capacity, growth kinetics and culture requirements for optimal proliferation. The primary cultures from both cells tended to be heterogeneous. Fibroblastic cells became progressively dominant with advancing passages. Similar to MSCs blastema passaged-3 cells succeeded to differentiate into bone, cartilage and adipose cell lineages. Even lineage specific genes tended to express in higher level in blastema cells compared to MSCs (p < 0.05). Moreover blastema cells appeared more proliferative; producing more colonies (p < 0.05). While blastema cells showed extensive proliferation in 15% fetal bovine serum (FBS), MSCs displayed higher expansion rate at 10% FBS. In conclusion, blastema from rabbit ear contains a population of fibroblastic cells much similar in characteristic to bone marrow mesenchymal stem cells. However, the two cells were different in the level of lineage-specific gene expression, the growth curve characteristics and the culture requirements.