Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed

Temporo-spatial distribution of blood vessels in human lumbar intervertebral discs

While there is consensus in the literature that blood vessels are confined to the outer anulus fibrosus of normal adult intervertebral disc, debate continues whether there is a vascular in-growths into inner parts of the intervertebral disc during degeneration. We therefore tested the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. The specific endothelial cell marker CD 31 (PECAM) was used to immunohistochemically investigate 42 paraffin-embedded complete mid-sagittal human intervertebral disc sections of various ages (0–86 years) and varying extent of histomorphological degeneration. Additionally, 20 surgical disc samples from individuals (26–69 years) were included in this study. In discs of fetal to infantile age, blood vessels perforated the cartilaginous end plate and extended into the inner and outer anulus fibrosus, but not into the nucleus pulposus. In adolescents and adults, no blood vessels were seen except for the outer zone of the anulus fibrosus adjacent to the insertion to ligaments. The cartilaginous end plate remained free of vessels, except for areas with circumscribed destruction of the end plate. In advanced disc degeneration, no vessels were observed except for those few cases with complete, scar-like disc destruction. However, some rim lesions and occasionally major clefts were surrounded by a small network of capillary blood vessels extending into deeper zones of the anulus fibrosus. A subsequent morphometric analysis, revealed slightly “deeper” blood vessel extension in juvenile/adolescent discs when compared to young, mature and senile adult individuals with significantly “deeper” extension in the posterior than anterior anulus. The analysis of the surgical specimens showed that only sparse capillary blood vessels which did not extend into the nucleus pulposus even in major disc disruption. Our results show that vascular invasion deeper than the periphery was not observed during disc degeneration, which supports the hypothesis that vascular in-growth is not a distinct feature of disc degeneration

Confiabilidade de um método de análise histológica da degeneração discal experimental em coelhos Confiabilidad de un método para el análisis histológico de la degeneración discal experimental en conejos Reliability of a method for histological analysis of experimental disc degeneration in rabbits

OBJETIVO: Validar um método para avaliação histológica da degeneração discal experimental em coelhos, baseando-se na confiabilidade da análise interobservador. MÉTODOS: Treze coelhos da raça New Zealand foram submetidos a procedimento de indução da degeneração discal através de punção direta de três discos intervertebrais (DIV) consecutivos com agulha 18G sob anestesia. Ao fim de dois meses, coletou-se a coluna vertebral completa de cada coelho e procedeu-se preparo das peças para análise histológica dos discos intervertebrais (Experimentais e Controle), sendo coradas pelo método hematoxilina-eosina. As lâminas histológicas foram avaliadas quanto à ocorrência de degeneração através da análise dos seguintes critérios: presença de vasos sanguíneos; presença de protrusão do núcleo pulposo (NP) através do ânulo fibroso (AF); e ruptura das fibras do AF. RESULTADOS: Ao final dos processos de eutanásia, retirada da coluna e preparo histológico, obteve-se 60 DIV viáveis para avaliação de degeneração. Deste total, 25 peças eram de DIV experimentais e 35 peças de DIV controle. A presença de vaso sanguíneo foi observada em 18 dos 25 DIV degenerados, com concordância de Kappa = 0,95 entre os observadores. A presença da extrusão do NP foi identificada em 19 dos 25 DIV experimentais, com concordância de Kappa = 0,78 entre os observadores. Com relação à ruptura das fibras do AF, pode-se identificar a positividade em 24 dos 25 DIV degenerados, com concordância entre os observadores de Kappa = 0,65. CONCLUSÃO: Este modelo de avaliação histológica da degeneração experimental do DIV mostrou-se viável, com alto grau de concordância entre os observadores na identificação da degeneração discal.<br>OBJETIVO: Validar un método para evaluación histológica de la degeneración discal experimental en conejos, basándose en la confiabilidad del análisis interobservadores. MÉTODOS: Trece conejos de la raza New Zealand fueron sometidos a procedimiento de inducción de la degeneración discal a través de punción directa de tres discos intervertebrales (DIV) consecutivos con aguja 18G bajo anestesia. Al fin de dos meses, se colectó la columna vertebral completa de cada conejo y se procedió a la preparación de las piezas para análisis histológico de los discos intervertebrales (Experimentales y Controles), siendo coloreadas por el método hematoxilina-eosina. Las láminas histológicas fueron evaluadas cuanto a ocurrencia de degeneración a través del análisis de los siguientes criterios: presencia de vasos sanguíneos; presencia de protrusión del núcleo pulposo (NP) a través del anillo fibroso (AF); y rotura de las fibras del AF. RESULTADOS: Al final de los procesos de eutanasia, remoción y preparación histológica de la columna, se obtuvieron 60 DIV viables para evaluación de degeneración. De este total, 25 piezas eran de DIV experimentales y 35 piezas de DIV control. La presencia de vaso sanguíneo fue observada en 18 de los 25 DIV degenerados, con concordancia de Kappa = 0,95 entre los observadores. La presencia de la extrusión del NP fue identificada en 19 de los 25 DIV experimentales, con concordancia de Kappa = 0,78 entre los observadores. Con relación a la rotura de las fibras del AF, se puede identificar la positividad en 24 de los 25 DIV degenerados, con concordancia entre los observadores de Kappa = 0,65. CONCLUSIÓN: Este modelo de evaluación histológica de la degeneración experimental del DIV se mostró viable, con alto grado de concordancia entre los observadores en la identificación de la degeneración discal.<br>OBJECTIVE: To validate a method for histological evaluation of disc degeneration in rabbits based on the reliability of interobserver analysis. METHODS: Thirteen New Zealand white rabbits underwent a procedure to induce disc degeneration through direct puncture of three consecutive intervertebral discs (IVD) with an 18G needle under anesthesia. After two months, the complete spine of each rabbit was collected, and further prepared for histologic examination of the intervertebral discs (Experimental and Control), and stained with the hematoxylin-eosin method. Histology slides were evaluated for the occurrence of degeneration by analyzing the following criteria: presence of blood vessel, presence of protrusion of the nucleus pulposus (NP) through the annulus fibrosus (AF), and rupture of the fibers of the AF. RESULTS: After the euthanasia procedures, collection of the spine and histological preparation, 60 viable IVD were obtained for degeneration assessment. Of a total of 60 pieces, 25 pieces were from Experimental IVD and 35 pieces were from Control IVD. The presence of blood vessel was observed in 18 of 25 degenerated IVD with agreement among observers of Kappa = 0.95. The presence of the extrusion of the NP was identified in 19 of 25 experimental IVD with interobserver agreement of Kappa = 0.78. The rupture of the fibers of AF could be identified in 24 of 25 degenerated IVD, with interobserver agreement of Kappa = 0.65. CONCLUSION: This model of histological assessment of experimental IVD degeneration is feasible, with a high degree of interobserver agreement in the identification of disc degeneration

Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc

The fate of notochord cells during disc development and aging is still a subject of debate. Cells with the typical notochordal morphology disappear from the disc within the first decade of life. However, the pure morphologic differentiation of notochordal from non-notochordal disc cells can be difficult, prompting the use of cellular markers. Previous reports on these notochordal cell markers only explored the occurrence in young age groups without considering changes during disc degeneration. The aim of this study, therefore, was to investigate presence, localization, and abundance of cells expressing notochordal cell markers in human lumbar discs during disc development and degeneration. Based on pilot studies, cytokeratins CK-8, -18 and -19 as well as Galectin-3 were chosen from a broad panel of potential notochordal cell markers and used for immunohistochemical staining of 30 human lumbar autopsy samples (0–86 years) and 38 human surgical disc samples (26–69 years). In the autopsy group, 80% of fetal to adolescent discs (0–17 years) and 100% of young adult discs (18–30 years) contained many cells with positive labeling. These cells were strongly clustered and nearly exclusively located in areas with granular changes (or other matrix defects), showing predominantly a chondrocytic morphology as well as (in a much lesser extent) a fibrocytic phenotype. In mature discs (31–60 years) and elderly discs (≥60 years) only 25 and 22–33%, respectively, contained few stained nuclear cells, mostly associated with matrix defects. In the surgical group, only 16% of samples from young adults (≤47 years) exhibited positively labeled cells whereas mature to old surgical discs (>47 years) contained no labeled cells. This is the first study describing the presence and temporo-spatial localization of cells expressing notochordal cell markers in human lumbar intervertebral discs of all ages and variable degree of disc degeneration. Our findings indicate that cells with a (immunohistochemically) notochord-like phenotype are present in a considerable fraction of adult lumbar intervertebral discs. The presence of these cells is associated with distinct features of (early) age-related disc degeneration, particularly with granular matrix changes

Clinical experience in cell-based therapeutics: intervention and outcome

Disc herniation treated by discectomy results in a significant loss of nucleus material and disc height. Biological restoration through the use of autologous disc chondrocyte transplantation (ADCT) offers a potential to achieve functional integration of disc metabolism and mechanics. Nucleus regeneration using autologous cultured disc-derived chondrocytes has been demonstrated in a canine model and in clinical pilot studies. In 2002 a prospective, controlled, randomized, multicentre study comparing safety and efficacy of ADCT plus discectomy, with discectomy alone was initiated. The clinical goals were to provide long-term pain relief, maintain disc height, and prevent adjacent segment disease. Interim analysis was performed after 2 years; Oswestry (Low Back Pain/disability), Quebec Back Pain Disability Scale, as well as Prolo and VAS Score were used for the evaluation. Disc height was assessed by MRI. A clinically significant reduction of low back pain in the ADCT-treated group was shown by all three pain score systems. The median total Oswestry Score was 2 in the ADCT group compared with 6 in the control group. Decreases in the Disability index in ADCT-treated patients correlated with the reduction of low back pain. Decreases in disc height over time were only found in the control group, and of potential significance, intervertebral discs in adjacent segments appeared to retain hydration when compared to those adjacent to levels that had undergone discectomy without cell intervention