Intervertebral disc (IVD) degeneration is etiologically associated with low back pain and is currently only treated in severe cases with spinal fusion. Regenerative medicine attempts to restore degenerated tissue by means of cells, hydrogels, and/or growth factors and can therefore be used to slow, halt, or reverse the degeneration of the IVD in a minimally invasive manner. Previously, the growth factors bone morphogenetic proteins 2 and 7 (BMP-2, -7) were shown to enhance disc regeneration, in vitro and in vivo. Since BMPs have only a short in vivo half-life, and to prevent heterotopic ossification, we evaluated the use of a slow release system for BMP-2 homodimers and BMP-2/7 heterodimers for IVD regeneration. BMP growth factors were conjugated to a fibrin/hyaluronic acid (FB/HA) hydrogel and intradiscally injected in a goat model of mild IVD degeneration to study safety and efficacy. Mild degeneration was induced in five lumbar discs of seven adult Dutch milk goats, by injections with the enzyme chondroitinase ABC. After 12 weeks, discs were treated with either FB/HA-hydrogel only or supplemented with 1 or 5 μg/mL of BMP-2 or BMP-2/7. BMPs were linked to the FB/HA hydrogels using a transglutaminase moiety, to be released through an incorporated plasmin cleavage site. After another 12 weeks, goats were sacrificed and discs were assessed using radiography, MRI T2* mapping, and biochemical and histological analyses. All animals maintained weight throughout the study and no heterotopic bone formation or other adverse effects were noted during follow-up. Radiographs showed significant disc height loss upon induction of mild degeneration. MRI T2* mapping showed strong and significant correlations with biochemistry and histology as shown before. Surprisingly, no differences could be demonstrated in any parameter between intervention groups. To our knowledge, this is the first large animal study evaluating BMPs conjugated to an FB/HA-hydrogel for the treatment of mild IVD degeneration. The conjugated BMP-2 and BMP-2/7 appeared safe, but no disc regeneration was observed. Possible explanations include too low dosages, short follow-up time, and/or insufficient release of the conjugated BMPs. These aspects should be addressed in future studies.

T2* mapping is an MRI technique for IVD evaluation which allows for measurements on a continuous scale thus minimising observer bias compared to grading systems. Although limited by a small sample size, this study showed a relatively good and linear correlation between T2* relaxation time and accepted parameters of disc degeneration. This suggests that T2* mapping is a promising tool to assess disc degeneration in clinical practice.

Biomechanical properties of healthy and degenerated nucleus pulposus (NP) are thought to be important for future regenerative strategies for intervertebral disc (IVD) repair. However, which properties are pivotal as design criteria when developing NP replacement materials is ill understood. Therefore, we determined and compared segmental biomechanics and NP viscoelastic properties in normal and mildly degenerated discs. In eight goats, three lumbar IVDs were chemically degenerated using chondroitinase ABC (CABC), confirmed with radiography and MRI after euthanasia 12 weeks post-operative. Neutral zone (NZ) stiffness and range of motion (ROM) were determined sagitally, laterally, and rotationally for each spinal motion segment (SMS) using a mechanical testing device. NPs were isolated for oscillatory shear experiments; elastic and viscous shear moduli followed from the ratio between shear stress and strain. Water content was quantified by weighing before and after freeze-drying. Disc height on radiographs and signal intensity on MRI decreased (6% and 22%, respectively, p < 0.01) after CABC treatment, confirming that chemical degeneration provides a good model of disc degeneration. Furthermore, CABC-injected IVDs had significantly lower NZ stiffness and larger ROM in lateral bending (LB) and axial rotation (AR) than controls. Rheometry consistently revealed significantly lower (10-12%) viscoelastic moduli after mild degeneration within goats, though the inter-animal differences were relatively large (complex modulus $12 to 41 kPa). Relative water content in the NP was unaffected by CABC, remaining at $75%. These observations suggest that viscoelastic properties have a marginal influence on mechanical behavior of the whole SMS. Therefore, when developing replacement materials the focus should be on other design criteria, such as biochemical cues and swelling pressure.

Traditionally tissue samples are analysed using protein or enzyme specific stains on serial sections to build up a picture of the distribution of components contained within them. In this study we investigated the potential of multivariate curve resolution-alternating least squares (MCR-ALS) to deconvolute 2nd derivative spectra of Fourier transform infrared (FTIR) microscopic images measured in transflectance mode of goat and human paraffin embedded intervertebral disc (IVD) tissue sections, to see if this methodology can provide analogous information to that provided by immunohistochemical stains and bioassays but from a single section. MCR-ALS analysis of non-degenerate and enzymatically in vivo degenerated goat IVDs reveals five matrix components displaying distribution maps matching histological stains for collagen, elastin and proteoglycan (PG), as well as immunohistochemical stains for collagen type I and II. Interestingly, two components exhibiting characteristic spectral and distribution profiles of proteoglycans were found, and relative component/tissue maps of these components (labelled PG1 and PG2) showed distinct distributions in non-degenerate versus mildly degenerate goat samples. MCR-ALS analysis of human IVD sections resulted in comparable spectral profiles to those observed in the goat samples, highlighting the inter species transferability of the presented methodology. Multivariate FTIR image analysis of a set of 43 goat IVD sections allowed the extraction of semi-quantitative information from component/tissue gradients taken across the IVD width of collagen type I, collagen type II, PG1 and PG2. Regional component/tissue parameters were calculated and significant correlations were found between histological grades of degeneration and PG parameters (PG1: p = 0.0003, PG2: p < 0.0001); glycosaminoglycan (GAG) content and PGs (PG1: p = 0.0055, PG2: p = 0.0001); and MRI T2* measurements and PGs (PG1: p = 0.0021, PG2: p < 0.0001). Additionally, component/tissue parameters for collagen type I and II showed significant correlations with total collagen content (p = 0.0204, p = 0.0127). In conclusion, the presented findings illustrate, that the described multivariate FTIR imaging approach affords the necessary chemical specificity to be considered an important tool in the study of IVD degeneration in goat and human IVDs.