Pieter-Paul A. Vergroesen

The Use of Poly(L-lactide-co-caprolactone) as a Scaffold for Adipose Stem Cells in Bone Tissue Engineering: Application in a Spinal Fusion Model

Since the early 1990s, tissue engineering has been heralded as a strategy that may solve problems associated with bone grafting procedures. The original concept of growing bone in the laboratory, however, has proven illusive due to biological, logistic, and regulatory problems. Fat-derived stem cells and synthetic polymers open new, more practicable routes for bone tissue engineering. In this paper, we highlight the potential of poly(L-lactide-co-caprolactone) (PLCL) to serve as a radiolucent scaffold in bone tissue engineering. It appears that PLCL quickly and preferentially binds adipose stem cells (ASCs), which proliferate rapidly and eventually differentiate into the osteogenic phenotype. An in vivo spinal fusion study in a goat model provides a preclinical proof-of-concept for a one-step surgical procedure with ASCs in bone tissue engineering.

A Biodegradable Glue for Annulus Closure: Evaluation of Strength and Endurance

Study Design. A biodegradable glue was biomechanically tested for annulus closure using nondegenerated goat intervertebral discs. Ultimate strength and endurance tests were performed using native and punctured discs as positive and negative controls, respectively. Objective. The aim of this study was to investigate the feasibility and biomechanical properties of a biodegradable glue for annulus closure. Summary of Background Data. There is an unmet clinical need for annulus closure techniques. Isocyanate-terminated tissue glues show potential because they adhere to annulus tissue, have an elastic modulus similar to the annulus, and show limited cytotoxicity to human annulus fibrosus cells. Methods. Three biomechanical tests were performed divided in 2 parts: part 1: ultimate strength tests comparing native, punctured (2.4-mm needle), and glued caprine intervertebral discs (n = 11 per group); part 2: 10 discs per group were subjected to a 10-day ex vivo endurance test of 864,000 load cycles, followed by ultimate strength tests. Outcome parameters include the restoration of strength after puncture, reduction of herniation in the endurance test, and conservation of glue strength after endurance testing. Results. Part 1: The glue partially restored subsidence to failure and yield strength/ultimate strength ratio of intervertebral discs. Part 2: During endurance testing, 40% of punctured discs failed compared with none of the glued discs. Endurance testing did not affect glue strength, and pooling of ultimate strength tests showed that the glue restored ultimate strength, work to failure, and yield strength/ultimate strength to 79%, 75%, and 119% of native values, respectively. Conclusion. A biodegradable isocyanate-terminated glue increases the force at which nucleus protrusion occurs, and it limits herniations during endurance or ultimate strength tests. Biomechanical tests in a bioreactor provide a low-cost assessment for annulus repair strategies; however, the clinical efficacy needs to be further addressed using long-term in vivo studies. Level of Evidence: N/A

Intradiscal pressure measurements: A challenge or a routine?

Intradiscal pressure (IDP) is an essential biomechanical parameter and has been the subject of numerous in vivo and in vitro investigations. Although currently available sensors differ in size and measurement principles, no data exist regarding inter-sensor reliability in measuring IDP. Moreover, although discs of various species vary significantly in size and mechanics, the possible effects of sensor insertion on the IDP have never been investigated. The present in vitro study aimed to address these issues. The synchronized signals of two differently sized pressure transducers (Ø1.33 and Ø0.36 mm) obtained during the measurements in two species (bovine and caprine) and their influence on the measured pressure were compared. First, the discs were subjected to three loading periods, and the pressure was measured simultaneously to assess the inter-sensor reliability. In the second test, the effect of the sensor size was evaluated by alternatingly inserting one transducer into the disc while recording the resulting pressure change with the second transducer. Although both sensors yielded similar pressure values (ICC: consistency: 0.964-0.999; absolute agreement: 0.845-0.996) when used simultaneously, the sensor size was determined to influence the measured pressure during the insertion tests. The magnitude of the effect differed between species; it was insignificant in the bovine specimens but significant in the caprine specimens, with a pressure increase of 0.31-0.64 MPa (median: 0.43 MPa) obtained when the larger sensor was inserted. The results suggest that sensor selection for IDP measurements requires special attention and can be crucial for species with smaller disc sizes.

The poro-elastic behaviour of the intervertebral disc: A new perspective on diurnal fluid flow

Diurnal disc height changes, due to fluid in- and outflow, are in equilibrium while daytime spinal loading is twice as long as night time rest. A direction-dependent permeability of the endplates, favouring inflow over outflow, reportedly explains this; however, fluid flow through the annulus fibrosus should be considered. This study investigates the fluid flow of entire intervertebral discs. Caprine discs were preloaded in saline for 24h under four levels of static load. Under sustained load, we modulated the disc׳s swelling pressure by exchanging saline for demineralised water (inflow) and back to saline (outflow), both for 24h. We measured disc height creep and used stretched exponential models to determine time-constants. During inflow disc height increased in relation to applied load, and during outflow disc height decreased to preload levels. When comparing in- and outflow phases, there was no difference in creep, and time-constants were similar indicating no direction-dependent resistance to fluid flow in the entire intervertebral disc. Results provoked a new hypothesis for diurnal fluid flow: in vitro time-constants for loading are shorter than for unloading and in vivo daytime loading is twice as long as night time unloading, i.e. in diurnal loading the intervertebral disc is closer to loading equilibrium than to unloading equilibrium. Per definition, fluid flow is slower close to equilibrium than far from equilibrium; therefore, as diurnal loading occurs closer to loading equilibrium, fluid inflow during night time unloading can balance fluid outflow during daytime loading, despite a longer time-constant.

The Effects of Single-Level Instrumented Lumbar Laminectomy on Adjacent Spinal Biomechanics

Study Design Biomechanical study. Objective Posterior instrumentation is used to stabilize the spine after a lumbar laminectomy. However, the effects on the adjacent segmental stability are unknown. Therefore, we studied the range of motion (ROM) and stiffness of treated lumbar spinal segments and cranial segments after a laminectomy and after posterior instrumentation in flexion and extension (FE), lateral bending (LB), and axial rotation (AR). These outcomes might help to better understand adjacent segment disease (ASD), which is reported cranial to the level on which posterior instrumentation is applied. Methods We obtained 12 cadaveric human lumbar spines. Spines were axially loaded with 250 N for 1 hour. Thereafter, 10 consecutive load cycles (4 Nm) were applied in FE, LB, and AR. Subsequently, a laminectomy was performed either at L2 or at L4. Thereafter, load-deformation tests were repeated, after similar preloading. Finally, posterior instrumentation was added to the level treated with a laminectomy before testing was repeated. The ROM and stiffness of the treated, the cranial adjacent, and the control segments were calculated from the load-displacement data. Repeated-measures analyses of variance used the spinal level as the between-subject factor and a laminectomy or instrumentation as the within-subject factors. Results After the laminectomy, the ROM increased (+19.4%) and the stiffness decreased (−18.0%) in AR. The ROM in AR of the adjacent segments also increased (+11.0%). The ROM of treated segments after instrumentation decreased in FE (−74.3%), LB (−71.6%), and AR (−59.8%). In the adjacent segments after instrumentation, only the ROM in LB was changed (−12.9%). Conclusions The present findings do not substantiate a biomechanical pathway toward or explanation for ASD.

Translational challenges for the development of a novel nucleus pulposus substitute: Experimental results from biomechanical and in vivo studies

Nucleus pulposus replacement therapy could offer a less invasive alternative to restore the function of moderately degenerated intervertebral discs than current potentially destructive surgical procedures. Numerous nucleus pulposus substitutes have already been investigated, to assess their applicability for intradiscal use. Still, the current choice of testing methods often does not lead to efficient translation into clinical application. In this paper, we present the evaluation of a novel nucleus pulposus substitute, consisting of a hydromed core and an electrospun envelope. We performed three mechanical evaluations and an in vivo pilot experiment. Initially, the swelling pressure of the implant was assessed in confined compression. Next, we incorporated the implant into mechanically damaged caprine lumbar intervertebral discs to determine biomechanical segment behaviour in bending and torsion. Subsequently, segments were serially tested in native, damaged and repaired conditions under dynamic axial compressive loading regimes in a loaded disc culture system. Finally, nucleus pulposus substitutes were implanted in a live goat spine using a transpedicular approach. In confined compression, nucleus pulposus samples as well as implants showed some load-bearing capacity, but the implant exhibited a much lower absolute pressure. In bending and torsion, we found that the nucleus pulposus substitute could partly restore the mechanical response of the disc. During dynamic axial compression in the loaded disc culture system, on the other hand, the implant was not able to recover axial compressive behaviour towards the healthy situation. Moreover, the nucleus pulposus substitutes did not remain in place in the in vivo situation but migrated out of the disc area. From these results, we conclude that implants may mimic native disc behaviour in simple mechanical tests, yet fail in other, more realistic set-ups. Therefore, we recommend that biomaterials for nucleus pulposus replacement be tested in testing modalities of increasing complexity and in their relevant anatomical surroundings, for a more reliable prediction of clinical potential.

Osteoarthritis and intervertebral disc degeneration: Quite different, quite similar

Intervertebral disc degeneration describes the vicious cycle of the deterioration of intervertebral discs and can eventually result in degenerative disc disease (DDD), which is accompanied by low‐back pain, the musculoskeletal disorder with the largest socioeconomic impact world‐wide. In more severe stages, intervertebral disc degeneration is accompanied by loss of joint space, subchondral sclerosis, and osteophytes, similar to osteoarthritis (OA) in the articular joint. Inspired by this resemblance, we investigated the analogy between human intervertebral discs and articular joints. Although embryonic origin and anatomy suggest substantial differences between the two types of joint, some features of cell physiology and extracellular matrix in the nucleus pulposus and articular cartilage share numerous parallels. Moreover, there are great similarities in the response to mechanical loading and the matrix‐degrading factors involved in the cascade of degeneration in both tissues. This suggests that the local environment of the cell is more important to its behavior than embryonic origin. Nevertheless, OA is widely regarded as a true disease, while intervertebral disc degeneration is often regarded as a radiological finding and DDD is undervalued as a cause of chronic low‐back pain by clinicians, patients and society. Emphasizing the similarities rather than the differences between the two diseases may create more awareness in the clinic, improve diagnostics in DDD, and provide cross‐fertilization of clinicians and scientists involved in both intervertebral disc degeneration and OA.

Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture.

Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges.