Helena Barreto Henriksson

Transplantation of human mesenchymal stems cells into intervertebral discs in a xenogeneic porcine model.

Study Design. Experimental and descriptive study of a xenotransplantation model in minipigs. Objective. To study survival and function of human mesenchymal stem cells (hMSCs) after transplantation into injured porcine spinal discs, as a model for cell therapy. Summary of Background Data. Biologic treatment options of the intervertebral disc are suggested for patients with chronic low back pain caused by disc degeneration. Methods. Three lumbar discs in each of 9 minipigs were injured by aspiration of the nucleus pulposus (NP), 2 weeks later hMSCs were injected in F12 media suspension (cell/med) or with a hydrogel carrier (Puramatrix) (cell/gel). The animals were sacrificed after 1, 3, or 6 months. Disc appearance was visualized by magnetic resonance imaging. Immunohistochemistry methods were used to detect hMSCs by antihuman nuclear antibody staining, and further performed for Collagen II, Aggrecan, and Collagen I. SOX 9, Aggrecan, Versican, Collagen IA, and Collagen IIA and Collagen IIB human mRNA expression was analyzed by real-time PCR. Results. At magnetic resonance imaging all injured discs demonstrated degenerative signs. Cell/gel discs showed fewer changes compared with cell/med discs and only injured discs at later time points. hMSCs were detected in 9 of 10 of the cell/gel discs and in 8 of 9 of the cell/med discs. Immunostaining for Aggrecan and Collagen type II expression were observed in NP after 3 and 6 months in gel/cell discs and colocalized with the antihuman nuclear antibody. mRNA expression of Collagen IIA, Collagen IIB, Versican, Collagen 1A, Aggrecan, and SOX9 were detected in both cell/med and cell/gel discs at the time points 3 and 6 months by real-time PCR. Conclusion. hMSCs survive in the porcine disc for at least 6 months and express typical chondrocyte markers suggesting differentiation toward disc-like cells. As in autologous animal models the combination with a three-dimensional-hydrogel carrier seems to facilitate differentiation and survival of MSCs in the disc. Xenotransplantation seems to be valuable in evaluating the possibility for human cell therapy treatment for intervertebral discs.

Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species.

Study Design. Descriptive experimental study in 4 different mammals. Objective. To investigate cell proliferation/regeneration and localize stem cells/progenitor cells within the intervertebral disc (IVD). Summary of Background Data. Disc degeneration (DD) is believed to play a major role in patients with chronic lumbar pain. Lately, biologic treatment options for DD have gained increasing interest. Normal regeneration processes within the IVD and have previously been sparsely described and therefore it is of great interest to increase the knowledge about these processes. Methods. Detection of cell proliferations zones and label-retaining cells were done by in vivo 5-bromo-2-deoxyuridine (BrdU) labeling in 18 rabbits, killed after 4, 6, 10, 14, 28, or 56 days. Results were visualized with immunohistochemistry and fluorescence/confocal microscopy. Localization of progenitor cell were further investigated by immunohistochemistry using antibodies towards Notch1, Delta4, Jagged1, C-KIT, KI67, and Stro-1 in normal IVD from rabbits (n = 3), rats (n = 2), minipigs (n = 2), and in human degenerated IVD (n = 4). Further, flowcytometry analysis using progenitor markers were performed on additional human IVD cells (n = 3). Results. BrdU positive cells were found in comparable numbers at early and late time points in most regions of the anulus fibrosus (AF) and nucleus pulposus demonstrating slow ongoing cell proliferation. In the AF border to ligament zone (AFo) and the perichondrium region (P) a stem cell niche-like pattern was determined (a high number of BrdU positive cells at early time points vs. only a few label retaining cells at later time points). In normal and DD tissue from the 4 investigated species progenitor cell markers were detected. Conclusion. The IVD is a tissue with ongoing slow cell proliferation both in the AF and the nucleus pulposus. The stem cell niche pattern detected in AFo and P can be suggested to play a role for IVD morphology and function. These findings may be of importance for the development of biologic treatment strategies.

Identification of a stem cell niche in the zone of Ranvier within the knee joint

A superficial lesion of the articular cartilage does not spontaneously self‐repair and has been suggested to be partly due to lack of progenitor cells within the joint that can reach the site of injury. To study whether progenitor cells are present within the joint, 3‐month‐old New Zealand white rabbits were exposed to bromodeoxyuridine (BrdU) for 12 consecutive days and were then sacrificed 4, 6, 10, 14, 28 and 56 days after the first BrdU administration. Presence of BrdU and localization of progenitor markers were detected using immunohistochemistry. After 10 days of BrdU exposure, BrdU‐positive cells, i.e. proliferating cells, were abundantly detected in the epiphyseal plate, the perichondrial groove of Ranvier, and in all zones of the articular cartilage. After a wash‐out period, BrdU‐positive cells were still present, i.e. those considered to be progenitor cells, in these regions of the knee except for the proliferative zone of the epiphyseal plate. Cells in the perichondrial groove of Ranvier were further positive for several markers associated with progenitor cells and stem cell niches, including Stro‐1, Jagged1, and BMPr1a. Our results demonstrate that a small population of progenitor cells is present in the perichondrial groove of Ranvier as well as within the articular cartilage in the knee. The perichondrial groove of Ranvier also demonstrates the properties of a stem cell niche.

Support of Concept That Migrating Progenitor Cells From Stem Cell Niches Contribute to Normal Regeneration of the Adult Mammal Intervertebral Disc: A Descriptive Study in the New Zealand White Rabbit

Study Design. Descriptive experimental study performed in rabbits of 2 age groups. Objective. To study and investigate presence of prechondrocytic cells and cell migration routes (MR) in the intervertebral disc (IVD) region to gain knowledge about the normal IVD regeneration pattern. Summary of Background Data. Disc degeneration is thought to play a major role in patients with chronic lumbar pain. Regeneration processes and cell migration within the IVD have been sparsely described. Therefore, it is of interest to increase knowledge of these processes in order to understand pathological conditions of the IVD. Methods. At the beginning of the experiment, 5-bromo-2-deoxyuridine (BrdU) in vivo labeling was performed in 2 groups of rabbits, 3 and 9 months old (total 27 rabbits). BrdU is incorporated into DNA during mitosis, and then it is gradually diluted with each cell division until it finally disappears. Incorporation of BrdU was then visualized by immunohistochemistry (IHC) at different time points providing cell division pattern and presence of slow-cycling cells in the IVD region. IVD tissue was investigated by IHC for growth and differentiation factor-5 (GDF5), SOX9 (chondrogenic lineage markers), SNAIL homolog 1 (SNAI1), SNAIL homolog 2 (SLUG) (migration markers), and &bgr;1-INTEGRIN (cellular adhesion marker). In addition, GDF5, SOX9, and BMPRIB expression were investigated on genetic level. Results. BrdU+ cells were observed in early time points in the IVD niche, adjacent to the epiphyseal plate, at later time points mainly in outer region of the annulus fibrosus for both age groups of rabbits, indicating a gradual migration of cells. The presence of SLUG, SNAI1, GDF5, SOX9, and &bgr;1-INTEGRIN was found in same regions. Conclusion. The results suggest a cellular MR from the IVD stem cell niche toward the annulus fibrosus and the inner parts of the IVD. These findings may be of importance for understanding IVD regenerative mechanisms and for future development of biological treatment strategies.

Premixed acidic calcium phosphate cement: Characterization of strength and microstructure

By using a premixed calcium phosphate cement (CPC), the handling properties of the cement are drastically improved, which is a challenge for traditional injectable CPCs. Previously premixed cements have been based on apatitic cements. In this article, acidic cement has been developed and evaluated. Monocalcium phosphate monohydrate and beta-tricalcium phosphate were mixed with glycerol to form a paste. As the paste does not contain water, no setting reaction starts and thus the working time is indefinite. Powder/liquid ratios (P/L) of 2.25, 3.5 and 4.75 were evaluated. Setting time (ST) and compressive strength (CS) were measured after 1 day, 1 week and 4 weeks in phosphate buffered saline (PBS) solution, and the corresponding microstructure was evaluated using electron microscopy and X-ray diffraction. The ST started when the cements were placed in PBS and ranged from 28 to 75 min, higher P/L gave a lower ST. Higher P/L also gave a higher CS, which ranged from 2 to 16 MPa. The microstructure mainly consisted of monetite, 1-5 microm in grain size. After 4 weeks in PBS, the strength increased. As acidic cements are resorbed faster in vivo, this cement should allow faster bone regeneration than apatitic cements. Premixed cements show a great handling benefit when compared with normal CPCs and can be formulated with similar ST and mechanical properties.

Investigation of different cell types and gel carriers for cell‐based intervertebral disc therapy, in vitro and in vivo studies

Biological treatment options for the repair of intervertebral disc damage have been suggested for patients with chronic low back pain. The aim of this study was to investigate possible cell types and gel carriers for use in the regenerative treatment of degenerative intervertebral discs (IVD). In vitro: human mesenchymal cells (hMSCs), IVD cells (hDCs), and chondrocytes (hCs) were cultivated in three gel types: hyaluronan gel (Durolane®), hydrogel (Puramatrix®), and tissue‐glue gel (TISSEEL®) in chondrogenic differentiation media for 9 days. Cell proliferation and proteoglycan accumulation were evaluated with microscopy and histology. In vivo: hMSCs or hCs and hyaluronan gel were co‐injected into injured IVDs of six minipigs. Animals were sacrificed at 3 or 6 months. Transplanted cells were traced with anti‐human antibodies. IVD appearance was visualized by MRI, immunohistochemistry, and histology. Hyaluronan gel induced the highest cell proliferation in vitro for all cell types. Xenotransplanted hMSCs and hCs survived in porcine IVDs for 6 months and produced collagen II in all six animals. Six months after transplantation of cell/gel, pronounced endplate changes indicating severe IVD degeneration were observed at MRI in 1/3 hC/gel, 1/3 hMSCs/gel and 1/3 gel only injected IVDs at MRI and 1/3 hMSC/gel, 3/3 hC/gel, 2/3 gel and 1/3 injured IVDs showed positive staining for bone mineralization. In 1 of 3 discs receiving hC/gel, in 1 of 3 receiving hMSCs/gel, and in 1 of 3 discs receiving gel alone. Injected IVDs on MRI results in 1 of 3 hMSC/gel, in 3 of 3 hC/gel, in 2 of 3 gel, and in 1 of 3 injured IVDs animals showed positive staining for bone mineralization. The investigated hyaluronan gel carrier is not suitable for use in cell therapy of injured/degenerated IVDs. The high cell proliferation observed in vitro in the hyaluronan could have been a negative factor in vivo, since most cell/gel transplanted IVDs showed degenerative changes at MRI and positive bone mineralization staining. However, this xenotransplantation model is valuable for evaluating possible cell therapy strategies for human degenerated IVDs. Copyright

Similar cellular migration patterns from niches in intervertebral disc and in knee-joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit.

IntroductionPotential stem cell niches (SNs) were recently reported in intervertebral discs (IVDs) and knee joints (KJs) in different mammals (located adjacent to the epiphyseal plate; EP). The aim here was to examine further possible cellular migration and migration directions of cells originating from niches possibly involved in regeneration of cartilaginous tissues in the IVD and in the KJ regions in adult mammals.MethodsIn total, 33 rabbits were used in studies A through C.A.IVD cells were sorted; fluorescence-activated cell sorting (FACS) by size (forward scatter; ≤10 μm or >10 μm or GDF5+ cells (anti-GDF5 antibody). Sorted cells, labeled with cell tracer (carboxyfluorescein-diacetate-succinimidyl ester; CDFA-SE) were applied on IVD explants in vitro. Migrating cells/distance was evaluated by fluorescence- and confocal-microscopy (FC).B.DNA labeling was performed with BrdU (oral administration). Animals were killed (14 to 56 days), KJs collected, and BrdU+ cells visualized with immunohistochemistry (IHC)/anti-BrdU antibody in SN and articular cartilage (AC).C.Cell tracer: (Fe-nanoparticles: Endorem) were injected into SNs of IVDs (LI-LV) and KJs (tibia). Animals were killed after 2 to 6 weeks. Fe-labeled cells were traced by ferric-iron staining (Prussian blue reaction; Mallory method).ResultsA. GDF5+ cells and ≤10-μm cells displayed the best migration capability in IVD explants. GDF5+ cells were detected at a tissue depth of 1,300 μm (16 days). B. BrdU+ cells were observed in early time points in niches of KJs, and at later time points in AC, indicating a gradual migration of cells. C. Fe+ cells were detected in IVDs; in annulus fibrosus (AF) in 11 of 12 animals and in nucleus pulposus (NP) in two of 12 animals. In AC (tibia), Fe+ cells were detected in six of 12 animals. In the potential migration route (PMR), from niches toward the IVD, Fe+ cells (three of 12 animals) and in PMR toward AC (KJs) (six of 12 animals) were detected.ConclusionsResults indicate similar cellular migration patterns in cartilage regions (IVD and KJs) with migration from stem cell niche areas into the mature cartilaginous tissues of both the KJs and the IVD. These findings of a cellular migration pattern in mature cartilage are of interest from tissue-repair and engineering perspectives.

Development and Regeneration Potential of the Mammalian Intervertebral Disc

At the present time, the normal cell proliferation rate and regeneration processes in the intervertebral disc (IVD) are not fully known. Historically, the IVD has been considered an organ with little or no regenerative capacity. However, several studies have identified the presence of cells expressing progenitor/stem cell markers in adult cartilage tissue and recent data suggest that adult mammalian IVDs have regenerative capacity, albeit slow. The aim of this review is to give an overview of the present knowledge regarding IVD development, regeneration and repair mechanisms in mammals, with a special focus on human discs. At a time when regenerative medicine is making progress and biological treatment options, such as stem cell therapy, are suggested for patients with degenerated discs causing chronic low back pain, basic knowledge about disc cells and their regenerative capacity form a useful basis for the exploration of new treatment options.

Physical Exercise Affects Cell Proliferation in Lumbar Intervertebral Disc Regions in Rats

Study Design. Descriptive experimental study. Objective. The aim of this study was to investigate the effect of exercise on cell proliferation in different areas of the intervertebral disc (IVD) and recruitment of cells possibly active in regeneration of normal rat lumbar IVDs. Summary of Background Data. Little is known about the effects of physical exercise on lumbar IVD tissue. Recently, stem cell niches in the perichondrium area of the IVD were identified and cells in these niches have been suggested to be involved in the normal regeneration of the IVD. Methods. Thirty Sprague-Dawley rats were exposed to 5-bromo-2-deoxyuridine (BrdU) diluted in the drinking water during 14 days. Fifteen rats ran on a treadmill daily for 50 min/d, 5 d/wk (exercise group), and 15 nonexercised rats served as controls. Immunohistochemical analyses (anti-BrdU antibody) were performed at 9, 14, 28, 56, and 105 days after the start of the exercise protocol. BrdU positive cells were counted in the stem cell niche area, the peripheral region of epiphyseal cartilage area, and the annulus fibrous outer and inner areas. Data were analyzed by 2-way analysis of variance (significance level; P < 0.05). Results. The BrdU positive cell numbers in the stem cell niche and annulus fibrous outer regions were increased in discs from the exercising group on days 14 (P < 0.01) and 105 (P < 0.05) and at day 14 (P < 0.01) in the peripheral epiphyseal cartilage region compared with controls. Conclusion. Physical exercise was shown to have positive effects on cell proliferation in IVDs, with involvement of various disc regions, indicating a differential response by disc tissue to exercise depending on anatomical location and tissue characteristics.

Biocompatibility and resorption of a radiopaque premixed calcium phosphate cement.

Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a Ø 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO(2) radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties.