Gianluca Vadalà

Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation

Recent studies have shown that mesenchymal stem cell (MSC)‐based therapy might be an effective approach for the treatment of intervertebral disc degeneration (IDD). However, many unanswered questions remain before clinical translation, such as the most effective stem cell type, a reliable transplantation method, including the carrier choice, and the fate of stem cells after misdirected delivery, among others. The objective of the study was to evaluate the fate and effect of allogenic bone marrow MSCs after transplantation into an IDD model. The L2–3, L3–4 and L4–5 intervertebral discs (IVDs) of four rabbits were stabbed to create IDD. Rabbit MSCs were expanded in vitro and in part transduced with retrovirus/eGFP. After 3 weeks, 1 × 105 MSCs were injected into the IVDs. The rabbits were followed by X‐ray and MRI 3 and 9 weeks after injection. Then the animals were sacrificed and the spines analysed histologically. MRI showed no signs of regeneration. X‐ray and gross anatomy inspection demonstrated large anterolateral osteophytes. Histological analysis showed that the osteophytes were composed of mineralized tissue surrounded by chondrocytes, with the labelled MSCs among the osteophyte‐forming cells. The labelled MSCs were not found in the nucleus. Inflammatory cells were not observed in any injected IVDs. These results raise concern that MSCs can migrate out of the nucleus and undesirable bone formation may occur. While cause cannot be inferred from this study, the presence of MSCs in the osteophytes suggests a potential side‐effect with this approach. IVD regeneration strategies need to focus on cell carrier systems and annulus‐sealing technologies to avoid pitfalls. Copyright

Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.

Study Design. Changes in gene expression profile and cell fusion of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) after coculture were analyzed. Objective. To investigate the mechanisms of the interaction between NPC and MSC such us differentiation, stimulatory effect, and cell fusion. Summary of Background Data. Introduction of exogenous cells to supplement and replenish intervertebral disc cell population offers a potential approach to treat intervertebral disc degeneration (IDD). Recent evidences showed that intradiscal injection of MSC effectively alter the course of IDD in vivo, and the regenerative potential may result from up-regulated extracellular matrix protein synthesis mediated by MSC and NPC interaction. Methods. Using a double labeling cell system and flow activated cell sorting, we quantitatively analyzed changes in the gene expression profile of human male MSC and female NPC after coculture in a 3-dimensional system that allows short distance paracrine interactions typical of the nucleus pulposus. Furthermore, we analyzed for cell fusion in the cell interaction by fluorescence in situ hybridization (FISH) for X and Y chromosomes, using a 3-dimensional culture system to allow cell-to-cell interactions conducive to cell fusion. Results. Two weeks of coculture cell interaction in a 3-dimensional environment induces a change in MSCs towards a more chondrogenic gene expression profile indicating MSC differentiation, and NPC gene expression changes in matrix and chondrogenic genes demonstrating only a modest trophic effect of MSC on NPC. Moreover, FISH analysis demonstrated that cell fusion is not responsible for MSC plasticity in the interaction with NPCs. Conclusion. This study clarifies the mechanism of MSCs and NPCs interaction in a 3-dimensional environment, excluding cell fusion. These data support the use of undifferentiated MSC for stem cell therapy for IDD treatment.

2D and 3D 3-tesla magnetic resonance imaging of the double bundle structure in anterior cruciate ligament anatomy

For anterior cruciate ligament (ACL) surgery using the anatomic approach of the double bundle concept it is helpful to describe the anteromedial (AM) and posterolateral (PL) bundle using Magnetic Resonance Imaging (MRI), since this is the most important preoperative parameter next to the physical examination. The aim of this study was to distinguish both bundles in MRI. In a prospective study we evaluated the double bundle structure in ACL anatomy with a 3-T ultra-high-field strength MR imaging of cadaver knees, which allows faster imaging times, increased resolution and increased signal-to-noise ratio. Using oblique sagittal and oblique coronal planes, we were able to distinguish the double bundle structure in each knee. The following arthroscopic evaluation of the knees confirmed our MRI findings. Our study demonstrates the possibility of distinguishing the two bundles in the native ACL with 3T MRI. Following examinations must study the value for clinical application by describing different rupture patterns of the bundles and correlating this to arthroscopy. It would be advantageous to know the rupture pattern in advance. Presurgical planning could be improved by reconstructing only the torn and preserving the intact bundle.

Use of an antifibrotic agent improves the effect of platelet-rich plasma on muscle healing after injury

BACKGROUND Muscle contusions are a common type of muscle injury and are frequently encountered in athletes and military personnel. Although these injuries are capable of healing in most instances, incomplete functional recovery often occurs because of the development of fibrosis in the muscle. We hypothesized that a combination of platelet-rich plasma (PRP) injection and oral administration of losartan (an antifibrotic agent) could enhance muscle healing by stimulating muscle regeneration and angiogenesis and by preventing fibrosis in contusion-injured skeletal muscle. METHODS Contusion injuries were created in the tibialis anterior muscles of mice. Two treatments were tested, alone and in combination: 20 μL of PRP injected into the contusion site one day after injury, and 10 mg/kg/day of losartan administered beginning three days after injury and continuing until the end point of the experiment. Muscle regeneration and fibrosis development were evaluated by histological analysis, and functional recovery was measured by physiological testing. RESULTS Muscle regeneration and muscle function were significantly promoted in the combined PRP + losartan treatment group compared with the other groups. Combined PRP + losartan treatment significantly decreased the expression of phosphorylated Smad2/3 and the development of fibrosis compared with PRP treatment alone, and it increased vascular endothelial growth factor (VEGF) expression and the number of CD31-positive structures compared with losartan treatment alone. Follistatin, a positive regulator of muscle growth, was expressed at a higher level in the PRP + losartan group compared with the other groups. CONCLUSIONS PRP + losartan combinatorial therapy improved overall skeletal muscle healing after muscle contusion injury by enhancing angiogenesis and follistatin expression and by reducing the expression of phosphorylated Smad2/3 and the development of fibrosis. These results suggest that blocking the expression of transforming growth factor (TGF)-β1 with losartan improves the effect of PRP therapy on muscle healing after a contusion injury. CLINICAL RELEVANCE These findings could contribute to the development of biological treatments that aid in the healing of skeletal muscle after injury.

Characterization of intervertebral disc aging: longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression.

Study Design. A cohort of young, healthy New Zealand White rabbits was followed longitudinally with serial magnetic resonance imaging (MRI) analysis and terminal analysis of histologic changes and gene expression. Objective. To examine the changes observed during normal aging in the intervertebral disc. Summary of Background Data. Although there is a correlation between aging and the onset of intervertebral disc degeneration (IDD), evidence suggests that distinct pathways are involved in these processes. Our group has characterized a reproducible rabbit model of IDD by MRI, radiograph, histology, and mRNA expression. However, no similar analysis has been performed longitudinally for intervertebral disc aging to allow comparison of these 2 important processes. Methods. Four skeletally mature female NZW rabbits were housed for 122 weeks, and lumbar spine MRIs were characterized serially. Histologic and quantitative gene expression analysis of the nucleus pulposus of these aging animals was performed, and compared with adult and young rabbits. Results. Mean MRI index decreased by <25% through 120 weeks. The histologic analysis showed changes in cell composition, with abundant notochordal cells in the young, chondrocyte-like cells and notochordal cells in the adult, and clusters of hypertrophic chondrocytes in the aging discs. The PCR analysis of the nucleus pulposus showed that gene expression of collagen decreased, whereas that for proteoglycans increased with aging. BMP-2, TIMP-1, and SOX-9 expression was significantly lower in the young compared with adult discs and TGF-&bgr;1 demonstrated lower gene expression in young and aging animals. Conclusion. Although dramatic cellular changes were observed, age-related MRI changes occurred in this rabbit model of normal aging at a much slower rate than in a previous injury model of degeneration. In addition, the gene expression analysis of the nucleus pulposus demonstrated remarkable differences between aging and injury induced degeneration. These results suggest that aging and injury contribute uniquely to the process of IDD.

T1ρ magnetic resonance imaging quantification of early lumbar intervertebral disc degeneration in healthy young adults.

Study Design. Cross-sectional study using T1&rgr; magnetic resonance imaging (MRI) of lumbar spine in healthy young adults. Objective. To evaluate early intervertebral disc degeneration (IDD) quantified by T1&rgr;- and T2-weighted MRI in asymptomatic young adults and to correlate T1&rgr; value with Pfirrmann degenerative grade, sex, and body mass index (BMI). Summary of Background Data. Intervertebral disc starts early to degenerate losing proteoglycan content in the nucleus pulposus (NP). A potential tool for the study of early stage of IDD is T1&rgr; MRI. T1&rgr; relaxation time of human discs has been correlated to proteoglycan content in previous studies. Methods. T1&rgr;- and T2-weighted images of the lumbar spine were obtained for 63 asymptomatic young subjects (34 men and 29 women; mean age, 22.95 ± 1.8 yr), with a 1.5-T MRI scanner. T1&rgr; mapping and values in the NP and anulus fibrosus (n = 315) were obtained. Degenerative grade was assessed using T2-weighted images, according to the Pfirrmann scale. Differences in T1&rgr; value between sexes, BMI, and linear regression analyses with degenerative grade were determined. Results. T1&rgr; values of NPs were significantly higher than those of anulus fibrosus at all levels. T1&rgr; values were significantly lower in women at L3–L4 and L4–L5 discs (P < 0.05). T1&rgr; values decreased linearly with degenerative grade. However, nondegenerated discs (Pfirrmann grades 1 and 2) showed a wide range of T1&rgr; relaxation time. No significant correlation was observed between T1&rgr; value and BMI. Conclusion. The data of this study showed a significant difference in IDD onset between sexes. T1&rgr; values correlate with Pfirrmann degenerative grade in young adults. However, the wide distribution of T1&rgr; values in healthy intervertebral disc highlights the low sensitivity of Pfirrmann grade to detect the early IDD changes. T1&rgr; can be potentially used as a clinical tool to identify early IDD and to create a reliable quantitative scale.

Effect of bupivacaine on intervertebral disc cell viability

BACKGROUND CONTEXT Bupivacaine is a local anesthetic commonly used to relieve or control pain in interventional spine procedures. Bupivacaine has been shown to be toxic to articular cartilage, which has similarities to intervertebral disc (IVD) cartilage, raising concern over a potentially negative effect of bupivacaine on the disc. PURPOSE To determine bupivacaines effect on cell viability of IVD cells in vitro and to elucidate whether this is through apoptosis or necrosis. STUDY DESIGN In vitro controlled study of bupivacaine effect on cell viability in human and rabbit IVD cells. SUBJECTS Rabbit annulus fibrosus (AF) tissue, nucleus pulposus (NP) cells, and knee articular chondrocytes were isolated from New Zealand white rabbits. Human AF and NP cells were isolated from stage 3 to 4 degenerative disc surgical specimens. OUTCOME MEASURES Cell viability was assessed after exposure to bupivacaine via trypan blue staining or flow cytometry. METHODS Annulus fibrosus and NP cells were grown in monolayer and alginate beads, respectively, to simulate their physiologic environment. The cells were then exposed to bupivacaine or saline control at 60 and 120 minutes and examined for cell viability. RESULTS Rabbit NP cell death demonstrated a time and dose dependence in response to bupivacaine. In addition, cell death was greater than that observed for articular chondrocytes. Rabbit AF tissue also demonstrated increased cell death in response to bupivacaine exposure. Human NP cells demonstrated time-dependent cell death, with greater necrosis than apoptosis. Annulus fibrosus cells grown in monolayers also resulted in similar effects, with greater necrosis rather than apoptosis. CONCLUSIONS Despite its pain relieving properties, bupivacaine decreases cell viability in rabbit and human disc cells in a time-dependent manner. In addition, the changes observed are greater than that seen for articular chondrocytes. This increase in cell death appears to be related to an increase in necrosis rather than apoptosis. Whether bupivacaine exerts similar effects in vivo or how this relates to overall clinical outcome remains to be explored.

3-T MR imaging of partial ACL tears: a cadaver study

Magnetic resonance imaging (MRI) is the most commonly used diagnostic imaging procedure for suspected injuries to the anterior cruciate ligament (ACL). However, MRI has less utility for the evaluation of partial ACL tears. The goal of this study was to evaluate the possibility of distinguishing partial ACL tears applying the double bundle concept by dividing the ACL anatomy in the anteromedial (AM) and posterolateral bundle (PL). Six human cadaver knees were used in this laboratory study. The protocol consisted of sagittal, oblique coronal, and oblique sagittal proton-density-weighted fast spin echo sequences. After MRI the AM and the PL bundle were severed to mimic different partial ACL rupture patterns. MRI scanning of each knee was repeated, to record the quantitative parameters tilt and ACL angles and discontinuity as a nonquantitative parameter. Three orthopaedic surgeons and two radiologists were enlisted as blinded observers to evaluate the images. The transection patterns could be differentiated by evaluating discontinuity both in the paracoronal and in the sagittal plane. Evaluating the transection patterns, the AM bundle reached a better result in both planes compared to the PL bundle and the paracoronal plane had a better result in assessing the transection patterns compared to the sagittal plane for the PL bundle. Partial ACL transections could predictably be recognized on oblique sagittal and oblique coronal planes utilizing 3-T MRI technology. This concept allows a more precise description of ACL rupture patterns and might lead to a more distinctive approach for reconstructive surgery. The presurgical planning could be improved by applying a treatment algorithm based on a description of each bundle as intact or ruptured, leading to a reconstruction of the torn and a preservation of the intact bundle.

Intervertebral disc regeneration: from the degenerative cascade to molecular therapy and tissue engineering.

Low back pain is one of the major health problems in industrialized countries, as a leading source of disability in the working population. Intervertebral disc degeneration has been identified as its main cause, being a progressive process mainly characterized by alteration of extracellular matrix composition and water content. Many factors are involved in the degenerative cascade, such as anabolism/catabolism imbalance, reduction of nutrition supply and progressive cell loss. Currently available treatments are symptomatic, and surgical procedures consisting of disc removal are often necessary. Recent advances in our understanding of intervertebral disc biology led to an increased interest in the development of novel biological treatments aimed at disc regeneration. Growth factors, gene therapy, stem cell transplantation and biomaterials‐based tissue engineering might support intervertebral disc regeneration by overcoming the limitation of the self‐renewal mechanism. The aim of this paper is to overview the literature discussing the current status of our knowledge from the degenerative cascade of the intervertebral disc to the latest molecular, cell‐based therapies and tissue‐engineering strategies for disc regeneration. Copyright

The transpedicular approach as an alternative route for intervertebral disc regeneration.

Study Design. Descriptive anatomical study on ovine and human cadaveric lumbar spinal segments. Objective. To describe the alternative transpedicular approach to deliver therapeutic agents into intervertebral disc (IVD). Summary of Background Data. The present delivery approach of therapeutic agents (growth factors/cells/hydrogels) within the IVD is through injection, via the annulus fibrosus (AF). However, it has recently been demonstrated that small needle puncture of the AF leads to further degeneration and disc herniation. In addition, the injected material has a high chance to be extruded through the AF injury. Methods. Lumbar ovine and human spinal segments were used. Under fluoroscopy, a 2-mm Kirschner wire was introduced in the caudal vertebra through the pedicle and the inferior endplate to the nucleus pulposus. Gross anatomy analysis and high-resolution peripheral quantitative computed tomography (HR-pQCT) were performed to assess the right position of the wire in pedicles. Discography and nucleotomy were performed using a 14G cannula insertion or a 2-mm arthroscopic shaver blade, respectively. Nucleoplasty was also performed with agarose gel/contrast agent and imaged with HR-pQCT. Results. Gross anatomy, fluoroscopy, and HR-pQCT images showed that the nucleus pulposus could be approached through the endplate via the pedicle without affecting the spinal canal and the neural foramina. The contrast agent was delivered into the IVD and nucleus pulposus was removed from the disc and filled with agarose gel. Conclusion. This study describes how a transpedicular approach can be used as an alternative route to deliver therapeutic agents to the disc without disruption of the AF showing the potential use of this technique in preclinical research and highlighting its clinical relevance for IVD regeneration.