Fabrizio Russo

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.

Stem cells sources for intervertebral disc regeneration

Intervertebral disc regeneration field is rapidly growing since disc disorders represent a major health problem in industrialized countries with very few possible treatments. Indeed, current available therapies are symptomatic, and surgical procedures consist in disc removal and spinal fusion, which is not immune to regardable concerns about possible comorbidities, cost-effectiveness, secondary risks and long-lasting outcomes. This review paper aims to share recent advances in stem cell therapy for the treatment of intervertebral disc degeneration. In literature the potential use of different adult stem cells for intervertebral disc regeneration has already been reported. Bone marrow mesenchymal stromal/stem cells, adipose tissue derived stem cells, synovial stem cells, muscle-derived stem cells, olfactory neural stem cells, induced pluripotent stem cells, hematopoietic stem cells, disc stem cells, and embryonic stem cells have been studied for this purpose either in vitro or in vivo. Moreover, several engineered carriers (e.g., hydrogels), characterized by full biocompatibility and prompt biodegradation, have been designed and combined with different stem cell types in order to optimize the local and controlled delivery of cellular substrates in situ. The paper overviews the literature discussing the current status of our knowledge of the different stem cells types used as a cell-based therapy for disc regeneration.

Spondylodiscitis: standards of current treatment.

Abstract Background: Spinal infections are an important clinical problem that often require aggressive medical therapy, and sometimes even surgery. Known risk factors are advanced age, diabetes mellitus, rheumatoid arthritis, immunosuppression, alcoholism, long-term steroid use, concomitant infections, poly-trauma, malignant tumor, and previous surgery or invasive procedures (discography, chemonucleolysis, and surgical procedures involving or adjacent to the intervertebral disc space). The most common level of involvement is at the lumbar spine, followed by the thoracic, cervical and sacral levels: lesions at the thoracic spine tend to lead more frequently to neurological symptoms. Objective: The aim of the current paper is to describe current evidence-based standards of therapy in the management of SD by emphasizing pharmacological therapy and principles and indications for bracing and surgery. Methods: A PubMed and Google Scholar search using various forms and combinations of the key words: spondylodiscitis, spine, infection, therapy, surgery, radiology, treatment. Reference citations from publications identified in the literature search were reviewed. Publications highlighted in this article were extracted based on relevancy to established, putative, and emerging diagnostic and therapeutic standards, either conservative (antibiotic therapy and bracing) or surgical. Findings: To date, conservative therapy, based on targeted antibiotic therapy plus bracing, represents the mainstay in the management of SD. Proper diagnosis and tailored therapy can improve clinical results and decrease the chance of failure. Surgery should be an option only for patients with complications of this disease, namely deformity, neural compression and neurological compromise. Current standards in the setting of SD are continuously evolving, as can be seen in the recent advances in the field of radiological diagnostics, and the use of growth factors and cell-therapy strategies to promote infection eradication and bone healing after surgery.

Platelet Rich Plasma and Hyaluronic Acid Blend for the Treatment of Osteoarthritis: Rheological and Biological Evaluation

Introduction Osteoarthritis (OA) is the most common musculoskeletal disease. Current treatments for OA are mainly symptomatic and inadequate since none results in restoration of fully functional cartilage. Hyaluronic Acid (HA) intra-articular injections are widely accepted for the treatment of pain associated to OA. The goal of HA viscosupplementation is to reduce pain and improve viscoelasticity of synovial fluid. Platelet-rich plasma (PRP) has been also employed to treat OA to possibly induce cartilage regeneration. The combination of HA and PRP could supply many advantages for tissue repair. Indeed, it conjugates HA viscosupplementation with PRP regenerative properties. The aim of this study was to evaluate the rheological and biological properties of different HA compositions in combination with PRP in order to identify (i) the viscoelastic features of the HA-PRP blends, (ii) their biological effect on osteoarthritic chondrocytes and (iii) HA formulations suitable for use in combination with PRP. Materials and Methods HA/PRP blends have been obtained mixing human PRP and three different HA at different concentrations: 1) Sinovial, 0.8% (SN); 2) Sinovial Forte 1.6% (SF); 3) Sinovial HL 3.2% (HL); 4) Hyalubrix 1.5% (HX). Combinations of phosphate buffered saline (PBS) and the four HA types were used as control. Rheological measurements were performed on an Anton PaarMCR-302 rheometer. Amplitude sweep, frequency sweep and rotational measurements were performed and viscoelastic properties were evaluated. The rheological data were validated performing the tests in presence of Bovine Serum Albumin (BSA) up to ultra-physiological concentration (7%). Primary osteoarthritic chondrocytes were cultured in vitro with the HA and PRP blends in the culture medium for one week. Cell viability, proliferation and glycosaminoglycan (GAG) content were assessed. Results PRP addition to HA leads to a decrease of viscoelastic shear moduli and increase of the crossover point, due to a pure dilution effect. For viscosupplements with HA concentration below 1% the viscoelasticity is mostly lost. Results were validated also in presence of proteins, which in synovial fluid are more abundant than HA. Chondrocytes proliferated overtime in all different culture conditions. The proliferation rate was higher in chondrocytes cultured in the media containing PRP compared to the cultures with different HA alone. GAG content was significantly higher in chondrocytes cultured in PRP and HL blend. Discussion We investigated the rheological and biological properties of four different HA concentrations when combined with PRP giving insights on viscoelastic and biological properties of a promising approach for future OA therapy. Our data demonstrate that PRP addition is not detrimental to the viscosupplementation effect of HA. Viscosupplements containing low HA concentration are not indicated for combination with PRP, as the viscoelastic properties are lost. Although having the same rheological behavior of SF and HX, HL was superior in stimulating extracellular matrix production in vitro.

A Nucleotomy Model with Intact Annulus Fibrosus to Test Intervertebral Disc Regeneration Strategies.

INTRODUCTION New cells/hydrogel-based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but does not allow the injection of a significant volume into intact IVD. The aim of this study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical nucleotomy (N), or a combining technique (E+N), as a model to study IVD regeneration strategies with intact annulus fibrosus (AF) in functional spinal units (FSUs) in vitro. METHODS The transpedicular approach via the endplate route (2 mm tunnel) was performed on ovine FSU (IVD and superior and inferior endplate) to access the NP. FSUs were treated by N (Arthroscopic shaver), E (Trypsin/Collagenase), or E+N. Treatments were evaluated macro- and microscopically. The degradation of proteoglycan (PG) around the cavity was assessed by gel electrophoresis. Cell viability was evaluated using the lactate dehydrogenase (LDH) assay. Cavity volume was quantified through computerized tomography after injection of agarose gel/contrast agent. RESULTS A cavity with intact AF was successfully created with all three methods. The N group showed high reproducibility, low PG degradation, and no endplate thinning. Histological analysis demonstrated NP matrix degradation in enzyme-treated groups, while the PG content was homogenous using mechanical discectomy. Cell viability was affected only in the E group. The cavity volume normalized to the total IVD volume was 5.2% ± 1.6% in E, 5.0% ± 1.4% in E+N, and 4.2% ± 0.1% in N. CONCLUSIONS Mechanical nucleotomy leads to a more reproducible and less destructive cavity in the NP. Enzymatic methods perform better in terms of cavity volume; however, the cells and PG of the surrounding tissue may be affected. The mechanical nucleotomy enables the creation of a cavity into the IVD while keeping the AF intact, allowing the injection of reproducible volumes of hydrogel and tissue engineering construct for preclinical tests.

Early intervertebral disc degeneration changes in asymptomatic weightlifters assessed by t1ρ-magnetic resonance imaging.

Study Design. Case-control study. Objective. To evaluate early intervertebral disc degeneration quantified by T1&rgr;- and T2-weighted magnetic resonance imaging (MRI) in asymptomatic weightlifters compared with a healthy control group matched for sex and age. Summary of Background Data. Athletes consistently recruit or transfer high levels of repetitive forces through the spine, and MRI has documented a higher rate of intervertebral disc degeneration in athletes compared with matched controls. This study aims to analyze the potential role of T1&rgr;-MRI in the assessment of early degenerative changes occurring in intervertebral discs of young asymptomatic weightlifters compared with healthy controls. Methods. Twenty-six asymptomatic young male weightlifters versus a sedentary control group matched for age and sex, both having no lower back pain nor any spinal symptoms, underwent MRI (1.5 T). Degenerative grade was assessed using T2-weighted images, according to the Pfirrmann scale. T1&rgr; mapping and values in the nucleus pulposus (n = 130) were obtained. Differences in T1&rgr; value between among the groups and linear regression analyses with degenerative grade were determined. Results. Pfirrmann degenerative grade did not show significant differences among groups. Instead, T1&rgr; values were significantly lower in the lumbar spine of weightlifters compared with controls (P < 0.05). T1&rgr; values decreased linearly with degenerative grade. Conclusion. T1&rgr; values were significantly lower in athletes compared with a sedentary matched control group showing differences in intervertebral disc degeneration onset among individuals with lifestyle and environmental factors leading to back pain. T1&rgr; can be potentially used as a valid clinical tool to identify early changes in intervertebral disc on the verge of new emerging intervertebral discs regenerative strategies and treatments. Level of Evidence: 4

Biomechanical Evaluation of Transpedicular Nucleotomy with Intact Annulus Fibrosus.

Study Design. Biomechanical testing of partially nucleotomized ovine cadaveric spines. Objective. To explore how the nucleus pulposus (NP) affects the biomechanical behavior of the intervertebral disc (IVD) by performing a partial nucleotomy via the transpedicular approach. Summary of Background Data. Mechanical loading represents a crucial part of IVD homeostasis. However, traditional regenerative strategies require violation of the annulus fibrosus (AF) resulting in significant alteration of joint mechanics. The transpedicular nucleotomy represents a suitable method to create a cavity into the NP, as a model to study IVD regeneration with intact AF. Methods. A total of 30 ovine-lumbar- functional spinal units (FSUs) (L1-L6) randomly assigned to 5 groups: control; transpedicular tunnel (TT); TT + polymethylmethacrylate (PMMA) to repair the bone tunnel; nucleotomy; nucleotomy + PMMA. Flexion/extension, lateral-bending, and axial-rotation were evaluated under adaptive displacement control. Axial compression was applied for 15 cycles of preconditioning followed by 1 hour of constant compression. Viscoelastic behavior was modeled and parameterized. Results. TT has minimal effects on rotational biomechanics. The nucleotomy increases ROM and neutral zone (NZ) displacement width whereas decreasing NZ stiffness. TT + PMMA has small effects in terms of ROM. Nucleotomy + PMMA brings ROM back to the control, increases NZ stiffness, and decreases NZ displacement width. The nucleotomy tends to increase the rate of early creep. TT reduces early and late damping. The use of PMMA increased late elastic stiffness (S2) and reduced viscous damping (&eegr;2) culminating in faster resolution of creep. Conclusion. Biomechanical properties of NP are crucial for IVD repair. This study demonstrated that TT does not affect rotational stability whereas partial nucleotomy with intact AF induce rotational instability, highlighting the central role of NP in early stages of IDD. Therefore, this model represents a successful platform to validate and optimize disc regeneration strategies. Level of Evidence: N/A

A clinically relevant hydrogel based on hyaluronic acid and platelet rich plasma as a carrier for mesenchymal stem cells: Rheological and biological characterization†

Intervertebral disc regeneration is quickly moving towards clinical applications. However, it is still missing an ideal injectable hydrogel to support mesenchymal stem cells (MSC) delivery. Herein, a new injectable hydrogel composed of platelet rich plasma (PRP) and hyaluronic acid (HA) blended with batroxobin (BTX) as gelling agent, was designed to generate a clinically relevant cell carrier for disc regeneration. PRP/HA/BTX blend was tested for rheological properties. Amplitude sweep, frequency sweep, and rotational measurements were performed and viscoelastic properties were evaluated. Human MSC encapsulated in PRP/HA/BTX hydrogel were cultured in both growing medium and medium with or without TGF‐β1 up to day 21. The amount of glycosaminoglycan was evaluated. Quantitative gene expression evaluation for collagen type II, aggrecan, and Sox 9 was also performed. Rheological tests showed that the hydrogel jellifies in 15 min 20°C and in 3 min at 37°C. Biological test showed that MSCs cultured in the hydrogel maintain high cell viability and proliferation. Human MSC within the hydrogel cultured with or without TGF‐β1 showed significantly higher GAG production compared to control medium. Moreover, MSCs in the hydrogel underwent differentiation to chondrocyte‐like cells with TGF‐β1, as shown by histology and gene expression analysis. This novel hydrogel improves viability and proliferation of MSCs supporting the differentiation process toward chondrocyte‐like cells. Rheology tests showed optimal gelation kinetics at room temperature for manipulation and faster gelation after transplantation (37°C). The clinical availability of all components of the hydrogel will allow a rapid translation of this regenerative approach into the clinical scenario.

How to treat lumbar disc herniation in pregnancy? A systematic review on current standards

PurposeIn this systematic review, we aim to illustrate the current and safe concepts in the assessment, diagnosis and management of herniated lumbar disc (HLD) during pregnancy.MethodsA systematic review and reporting on the diagnosis, treatment and clinical results of HLD during pregnancy is performed.ResultsThe MRI represents the first level and safest diagnostic tool for pregnant women affected by spinal problems allowing for a noninvasive and detailed radiological examination of the spine. The initial management of pregnant women affected by HLD is conservative, and primarily aimed to pain therapy. Whenever radicular pain and progressive neurological deficits unresponsive to medical management occur, surgery should be considered. Few case reports regarding the operative management of HLD in pregnant women have been published up to date. Laminectomy and/or microdiscectomy represent the classical and most commonly used techniques that can be safely performed without affecting pregnancy, delivery, or baby’s health. Endoscopic discectomy may be an alternative. The most adequate timing and surgical position are chosen based on to the fetal gestational age and site of the pathology.ConclusionsSurgical treatments during pregnancy impose multiple medical and ethical problems. Timely diagnosis by MRI, careful clinical evaluation, and surgical treatment represent safe and effective procedures. Ongoing evolution of surgical, anesthesiological and obstetrical procedures results in favorable outcomes. However, interdisciplinary management and a wide knowledge of pregnancy-related pathologies are crucial for the best outcome for both mother and child.