Publications by authors named "Mattie H P van Rijen"

24 Publications

  • Page 1 of 1

Sensory Innervation of Human Bone: An Immunohistochemical Study to Further Understand Bone Pain.

J Pain 2021 May 5. Epub 2021 May 5.

Department of Orthopedic Surgery, University Medical Center Utrecht, The Netherlands; SentryX B.V., Woudenbergseweg 41, Austerlitz, The Netherlands.

Skeletal diseases and their surgical treatment induce severe pain. The innervation density of bone potentially explains the severe pain reported. Animal studies concluded that sensory myelinated A∂-fibers and unmyelinated C-fibers are mainly responsible for conducting bone pain, and that the innervation density of these nerve fibers was highest in periosteum. However, literature regarding sensory innervation of human bone is scarce. This observational study aimed to quantify sensory nerve fiber density in periosteum, cortical bone, and bone marrow of axial and appendicular human bones using immunohistochemistry and confocal microscopy. Multivariate Poisson regression analysis demonstrated that the total number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow, and cortical bone for all bones studied. Bone from thoracic vertebral bodies contained most sensory nerve fibers, followed by the upper extremity, lower extremity, and parietal neurocranium. The number of nerve fibers declined with age and did not differ between male and female specimens. Sensory nerve fibers were organized as a branched network throughout the periosteum. The current results provide an explanation for the severe pain accompanying skeletal disease, fracture, or surgery. Further, the results could provide more insight into mechanisms that generate and maintain skeletal pain and might aid in developing new treatment strategies. PERSPECTIVE: This article presents the innervation of human bone and assesses the effect of age, gender, bone compartment and type of bone on innervation density. The presented data provide an explanation for the severity of bone pain arising from skeletal diseases and their surgical treatment.
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http://dx.doi.org/10.1016/j.jpain.2021.04.006DOI Listing
May 2021

Instrumented cervical fusion in nine dogs with caudal cervical spondylomyelopathy.

Vet Surg 2019 Oct 22;48(7):1287-1298. Epub 2019 Aug 22.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Objective: To report the long-term outcome of nine dogs treated for caudal cervical spondylomyelopathy (CCSM) with surgical spinal fusion.

Study Design: Short case series.

Animals: Nine large-breed dogs.

Methods: Medical records of dogs treated for disc-associated CCSM (2013-2016) were reviewed. The surgery objective was spinal distraction by implantation of a SynCage and fixation with two Unilock plates. Follow-up included the Helsinki pain score questionnaire, neurological grading, radiography, computed tomography (CT), and micro-CT (μCT) with subsequent histopathology (two dogs).

Results: Clinical follow-up was obtained between 9 and 51 months (27.4 ± 13.4 months). The Helsinki pain score and neurological Griffith score improved (P < .01) in all dogs and in eight of nine dogs, respectively. According to CT, the volume of bone (mean ± SD) through the cage was 79.5% ± 14.3%, including compact bone (53.0% ± 23.4%). Subsidence was seen in one of nine dogs. Implant failure was evident in four dogs, and plates were removed in two dogs. In seven of nine dogs, infraclinical pathology was observed in adjacent segment, associated with implants engaging adjacent intervertebral discs. Radiographic evidence of bony fusion between vertebral bodies was noted in all dogs. Spinal fusion was confirmed by μCT and histopathology in two cervical spine segments that became available at 22 and 40 months postoperatively.

Conclusion: Instrumented spinal fusion in dogs with disc-associated CCSM resulted in owner satisfaction and radiographic evidence of interbody spinal fusion in all dogs.

Clinical Significance: The fusion distraction technique reported here can be used to achieve spinal fusion with a good long-term outcome.
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http://dx.doi.org/10.1111/vsu.13312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790691PMC
October 2019

Comparing Hydrogels for Human Nucleus Pulposus Regeneration: Role of Osmolarity During Expansion.

Tissue Eng Part C Methods 2018 04 2;24(4):222-232. Epub 2018 Apr 2.

1 Department of Orthopedics, University Medical Center Utrecht , Utrecht, The Netherlands .

Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated NP have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation in an attempt to provide comparators for future studies. Human NP cells of Thompson grade III discs were cultured in alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid-poly(ethylene glycol) hydrogels. Medium containing fetal bovine serum and a serum-free (SF) medium were compared in agarose, gelMA, and type II collagen hydrogels. Isolation and expansion of NP cells in low compared to high osmolarity medium were performed before culture in agarose and type II collagen hydrogels in media of varying osmolarity. NP cells in agarose produced the highest amounts of proteoglycans, followed by cells in type II collagen hydrogels. The absence of serum reduced the total amount of proteoglycans produced by the cells, although incorporation efficiency was higher in type II collagen hydrogels in the absence than in the presence of serum. Isolation and expansion of NP cells in high osmolarity medium improved proteoglycan production during culture in hydrogels, but variation in osmolarity during redifferentiation did not have any effect. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because, as opposed to agarose, it degrades in time. Although culture in SF medium reduces the amount of proteoglycans produced during redifferentiation culture, isolating and expanding the cells in high osmolarity medium can largely compensate for this loss.
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http://dx.doi.org/10.1089/ten.TEC.2017.0226DOI Listing
April 2018

Focal adhesion signaling affects regeneration by human nucleus pulposus cells in collagen- but not carbohydrate-based hydrogels.

Acta Biomater 2018 01 23;66:238-247. Epub 2017 Nov 23.

Department of Orthopedics, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observedenhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix.

Statement Of Significance: Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.
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http://dx.doi.org/10.1016/j.actbio.2017.11.029DOI Listing
January 2018

Effects of non-enzymatic glycation on the micro- and nano-mechanics of articular cartilage.

J Mech Behav Biomed Mater 2018 01 30;77:551-556. Epub 2017 Sep 30.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands; Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), Mekelweg 2, Delft 2628 CD, The Netherlands; Department of Rheumatology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

The mechanical properties of articular cartilage depend on the quality of its matrix, which consists of collagens and glycosaminoglycans (GAGs). The accumulation of advanced glycation end products (AGEs) can greatly affect the mechanics of cartilage. In the current study, we simulated the accumulation of AGEs by using L-threose to cross-link collagen molecules in the cartilage matrix (in vitro). The resulting changes in the mechanical properties (stiffness) of cartilage are then measured both at the micrometer-scale (using micro-indenter) and nanometer-scale (using indentation-type atomic force microscopy). Non-enzymatic cross-linking within the cartilage matrix was confirmed by the browning of L-threose-treated samples. We observed > 3 times increase in the micro-scale stiffness and up to 12-fold increase in the nano-scale stiffness of the glycated cartilage in the peak pertaining to the collagen fibers, which is caused by cartilage network embrittlement. At the molecular level, we found that besides the collagen component, the glycation process also influenced the GAG macromolecules. Comparing cartilage samples before and after L-threose treatment revealed that artificially induced-AGEs also decelerate in vitro degradation (likely via matrix metalloproteinases), observed at both micro- and nano-scales. The combined observations suggest that non-enzymatic glycation may play multiple roles in mechanochemical functioning of articular cartilage.
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http://dx.doi.org/10.1016/j.jmbbm.2017.09.035DOI Listing
January 2018

Ex vivo model unravelling cell distribution effect in hydrogels for cartilage repair.

ALTEX 2018 8;35(1):65-76. Epub 2017 Sep 8.

Department of Oral and Maxillofacial Surgery & Special Dental Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

The implantation of chondrocyte-laden hydrogels is a promising cartilage repair strategy. Chondrocytes can be spatially positioned in hydrogels and thus in defects, while current clinical cell therapies introduce chondrocytes in the defect depth. The main aim of this study was to evaluate the effect of spatial chondrocyte distribution on the reparative process. To reduce animal experiments, an ex vivo osteochondral plug model was used and evaluated. The role of the delivered and endogenous cells in the repair process was investigated. Full thickness cartilage defects were created in equine osteochondral plugs. Defects were filled with (A) chondrocytes at the bottom of the defect, covered with a cell-free hydrogel, (B) chondrocytes homogeneously encapsulated in a hydrogel, and (C, D) combinations of A and B with different cell densities. Plugs were cultured for up to 57 days, after which the cartilage and repair tissues were characterized and compared to baseline samples. Additionally, at day 21, the origin of cells in the repair tissue was evaluated. Best outcomes were obtained with conditions C and D, which resulted in well-integrated cartilage-like tissue that completely filled the defect, regardless of the initial cell density. A critical role of the spatial chondrocyte distribution in the repair process was observed. Moreover, the osteochondral plugs stimulated cartilage formation in the hydrogels when cultured in the defects. The resulting repair tissue originated from the delivered cells. These findings confirm the potential of the osteochondral plug model for the optimization of the composition of cartilage implants and for studying repair mechanisms.
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http://dx.doi.org/10.14573/altex.1704171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116182PMC
August 2018

Fixation of Hydrogel Constructs for Cartilage Repair in the Equine Model: A Challenging Issue.

Tissue Eng Part C Methods 2017 11;23(11):804-814

1 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University , Utrecht, The Netherlands .

Objective: To report on the experiences with the use of commercial and autologous fibrin glue (AFG) and of an alternative method based on a 3D-printed polycaprolactone (PCL) anchor for the fixation of hydrogel-based scaffolds in an equine model for cartilage repair.

Methods: In a first study, three different hydrogel-based materials were orthotopically implanted in nine horses for 1-4 weeks in 6 mm diameter full-thickness cartilage defects in the medial femoral trochlear ridge and fixated with commercially available fibrin glue (CFG). One defect was filled with CFG only as a control. In a second study, CFG and AFG were compared in an ectopic equine model. The third study compared the efficacy of AFG and a 3D-printed PCL-based osteal anchor for fixation of PCL-reinforced hydrogels in three horses for 2 weeks, with a 4-week follow-up to evaluate integration of bone with the PCL anchor. Short-term scaffold integration and cell infiltration were evaluated by microcomputed tomography and histology as outcome parameters.

Results: The first study showed signs of subchondral bone resorption in all defects, including the controls filled with CFG only, with significant infiltration of neutrophils. Ectopically, CFG induced clear inflammation with strong neutrophil accumulation; AFG was less reactive, showing fibroblast infiltration only. In the third study the fixation potential for PCL-reinforced hydrogels of AFG was inferior to the PCL anchor. PCL reinforcement had disappeared from two defects and showed signs of dislodging in the remaining four. All six constructs fixated with the PCL anchor were still in place after 2 weeks. At 4 weeks, the PCL anchor showed good integration and signs of new bone formation.

Conclusions: The use of AFG should be preferred to xenogeneic products in the horse, but AFG is subject to individual variations and laborious to make. The PCL anchor provides the best fixation; however, this technique involves the whole osteochondral unit, which entails a different conceptual approach to cartilage repair.
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http://dx.doi.org/10.1089/ten.TEC.2017.0200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116030PMC
November 2017

Allogeneic MSCs and Recycled Autologous Chondrons Mixed in a One-Stage Cartilage Cell Transplantion: A First-in-Man Trial in 35 Patients.

Stem Cells 2017 08 23;35(8):1984-1993. Epub 2017 Jun 23.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

MSCs are known as multipotent mesenchymal stem cells that have been found capable of differentiating into various lineages including cartilage. However, recent studies suggest MSCs are pericytes that stimulate tissue repair through trophic signaling. Aimed at articular cartilage repair in a one-stage cell transplantation, this study provides first clinical evidence that MSCs stimulate autologous cartilage repair in the knee without engrafting in the host tissue. A phase I (first-in-man) clinical trial studied the one-stage application of allogeneic MSCs mixed with 10% or 20% recycled defect derived autologous chondrons for the treatment of cartilage defects in 35 patients. No treatment-related serious adverse events were found and statistically significant improvement in clinical outcome shown. Magnetic resonance imaging and second-look arthroscopies showed consistent newly formed cartilage tissue. A biopsy taken from the center of the repair tissue was found to have hyaline-like features with a high concentration of proteoglycans and type II collagen. DNA short tandem repeat analysis delivered unique proof that the regenerated tissue contained patient-DNA only. These findings support the hypothesis that allogeneic MSCs stimulate a regenerative host response. This first-in-man trial supports a paradigm shift in which MSCs are applied as augmentations or "signaling cells" rather than differentiating stem cells and opens doors for other applications. Stem Cells 2017;35:1984-1993.
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http://dx.doi.org/10.1002/stem.2657DOI Listing
August 2017

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.

Stem Cells 2017 01 29;35(1):256-264. Epub 2016 Aug 29.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264.
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http://dx.doi.org/10.1002/stem.2475DOI Listing
January 2017

Groove model of tibia-femoral osteoarthritis in the rat.

J Orthop Res 2017 03 29;35(3):496-505. Epub 2016 May 29.

Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, UMC Utrecht, F.02.127, P.O. Box 85500, 3508 GA Utrecht, The Netherlands.

Several experimental models of osteoarthritis in rats are used to study the pathophysiology of osteoarthritis. Many mechanically induced models have the limitation that permanent joint instability is induced by, for example, ligament transection or meniscal damage. This permanent instability will counteract the potential beneficial effects of therapy. The groove model of osteoarthritis uses a one-time trigger, surgically induced cartilage damage on the femoral condyles, and has been validated for the canine tibia-femoral compartment. The present study evaluates this model for the rat knee joint. The articular cartilage of the weight bearing surface of both femoral condyles and trochlea were damaged (grooved) without damaging the underlying subchondral bone. Severity of joint degeneration was histologically assessed, in addition to patella cartilage damage, and subchondral bone characteristics by means of (contrast-enhanced) micro-CT. Mild histological degeneration of the surgically untouched tibial plateau cartilage was observed in addition to damage of the femoral condyles, without clear synovial tissue inflammation. Contrast enhanced micro-CT demonstrated proteoglycan loss of the surgically untouched patella cartilage. Besides, a more sclerotic structure of the subchondral bone was observed. The tibia-femoral groove model in a rat results in mild knee joint degeneration, without permanent joint instability and joint inflammation. This makes the rat groove model a useful model to study the onset and progression of post-traumatic non-inflammatory osteoarthritis, creating a relatively sensitive model to study disease modifying osteoarthritic drugs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:496-505, 2017.
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http://dx.doi.org/10.1002/jor.23299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363339PMC
March 2017

Histological characteristics of diffuse idiopathic skeletal hyperostosis.

J Orthop Res 2017 01 29;35(1):140-146. Epub 2016 Apr 29.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

Diffuse idiopathic skeletal hyperostosis (DISH) is a predominantly radiographic diagnosis and histological knowledge of DISH is limited. The aim of this study was to describe the histological characteristics of DISH in the spinal column and to study the relation between DISH and intervertebral disc (IVD) degeneration. Therefore, 10 human cadaveric spines with fluoroscopic evidence of DISH were compared with 10 controls. Plain radiographs and computed tomography (CT) scans were obtained and tissue blocks were resected from three predefined levels of all specimens. The microscopic sections were scored by two blinded observers using a newly developed scoring system specific for characteristics of DISH and a validated scoring system for IVD degeneration. Maximum IVD height was measured on the CT scans. Analyses were performed using Fisher's exact test and Student's t-test. When compared to controls, the right sided sections from DISH specimens showed partial or complete bone bridges, consisting of cortical woven bone, accompanied by morphological changes in the adjoining part of the IVD. Using the histological scoring system for DISH, all parameters were significantly different between the DISH and control group (p < 0.01). The contralateral location did not show differences between the groups. The overall degree of IVD degeneration and height of IVD was comparable for the two groups. The histopathological changes observed in spines with DISH corresponded to the fluoroscopic images and CT scans. The degree of IVD degeneration and IVD height was comparable for both groups, suggesting a limited role for IVD degeneration in the pathogenesis of DISH. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:140-146, 2017.
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http://dx.doi.org/10.1002/jor.23267DOI Listing
January 2017

Direct Cell-Cell Contact with Chondrocytes Is a Key Mechanism in Multipotent Mesenchymal Stromal Cell-Mediated Chondrogenesis.

Tissue Eng Part A 2015 Oct 12;21(19-20):2536-47. Epub 2015 Aug 12.

1 Department of Orthopaedics, University Medical Center Utrecht , Utrecht, The Netherlands .

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.
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http://dx.doi.org/10.1089/ten.TEA.2014.0673DOI Listing
October 2015

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration.

Arthritis Res Ther 2015 May 27;17:137. Epub 2015 May 27.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Yalelaan 108, 3584 CM, Utrecht, The Netherlands.

Introduction: Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.

Methods: Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.

Results: In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.

Conclusions: An intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.
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http://dx.doi.org/10.1186/s13075-015-0625-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443547PMC
May 2015

Crosslinkable hydrogels derived from cartilage, meniscus, and tendon tissue.

Tissue Eng Part A 2015 Apr 9;21(7-8):1195-206. Epub 2015 Feb 9.

1 Department of Orthopaedics, University Medical Center Utrecht , Utrecht, The Netherlands .

Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.
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http://dx.doi.org/10.1089/ten.TEA.2014.0362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394887PMC
April 2015

Chondrogenic potential of articular chondrocytes depends on their original location.

Tissue Eng Part A 2014 Feb 14;20(3-4):663-71. Epub 2013 Nov 14.

1 Department of Orthopaedics, University Medical Center Utrecht , Utrecht, The Netherlands .

Objective: This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies.

Methods: Articular cartilage was obtained from Outerbridge grade III and IV cartilage lesions and from macroscopically healthy weight-bearing and nonweight-bearing (NWB) locations in the knee. Chondrocytes isolated from all locations were either pelleted directly (P0 pellets) or after expansion (P2 pellets) and analyzed for glycosaminoglycan (GAG), DNA, and cartilage-specific gene expression. Harvested cartilage samples and cultured pellets were also analyzed by Safranin O histology and immunohistochemistry for collagen I, II, and X. Immunohistochemical stainings were quantified using a computerized pixel-intensity staining segmentation method.

Results: After 4 weeks of culture, the P0 pellets derived from grade III or healthy weight-bearing chondrocytes contained more (p<0.015) GAG and GAG normalized per DNA compared to those from grade IV and NWB locations. After expansion, these differences were lost. Cartilage-specific gene expression was higher (p<0.04) in P0 pellets from grade III chondrocytes compared to grade IV chondrocytes. Semiquantitative immunohistochemistry showed a more intense (p<0.033) collagen I and X staining for grade IV debrided cartilage compared to grade III and weight-bearing cartilage. Also, collagen type X staining intensity was higher (p<0.033) in NWB cartilage compared to grade III and weight-bearing regions.

Conclusion: Chondrocytes derived from debrided cartilage perform better than cells from the NWB biopsy site, however, this difference is lost upon expansion. Based thereon, the debrided defect cartilage could be a viable donor site for regenerative cartilage surgery.
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http://dx.doi.org/10.1089/ten.TEA.2012.0673DOI Listing
February 2014

Single-stage cell-based cartilage regeneration using a combination of chondrons and mesenchymal stromal cells: comparison with microfracture.

Am J Sports Med 2013 Sep 5;41(9):2158-66. Epub 2013 Jul 5.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, the Netherlands.

Background: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix.

Purpose: To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models.

Study Design: Controlled laboratory study.

Methods: Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs.

Results: The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment.

Conclusion: The combination of chondrons and MSCs increased cartilage matrix formation, and this combination of cells was safely applied in a goat model for focal cartilage lesions, outperforming microfracture.

Clinical Relevance: This study describes the bench-to-preclinical development of a new cell-based regenerative treatment for focal articular cartilage defects that outperforms microfracture in goats. In addition, it is a single-step procedure, thereby making the expensive cell expansion and reimplantation of dedifferentiated cells, as in ACI, redundant.
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http://dx.doi.org/10.1177/0363546513494181DOI Listing
September 2013

Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

Tissue Eng Part A 2012 Oct 25;18(19-20):1957-66. Epub 2012 Jun 25.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well as for endochondral bone tissue engineering, as these approaches deal with, respectively, the inhibition or enhancement of hypertrophic chondrogenesis.
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http://dx.doi.org/10.1089/ten.TEA.2011.0657DOI Listing
October 2012

Identification of cell surface-specific markers to target human nucleus pulposus cells: expression of carbonic anhydrase XII varies with age and degeneration.

Arthritis Rheum 2011 Dec;63(12):3876-86

University College Dublin, Dublin, Ireland.

Objective: Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents.

Methods: We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study.

Results: Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue.

Conclusion: CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.
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http://dx.doi.org/10.1002/art.30607DOI Listing
December 2011

Hypoxia impedes vasculogenesis of in vitro engineered bone.

Tissue Eng Part A 2012 Jan 4;18(1-2):208-18. Epub 2011 Oct 4.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.
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http://dx.doi.org/10.1089/ten.TEA.2010.0731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246420PMC
January 2012

Replacement of the medial tibial plateau by a metallic implant in a goat model.

J Orthop Res 2010 Apr;28(4):429-35

Department of Orthopaedics, University Medical Center Utrecht, P.O. Box 85500 3508 GA Utrecht, The Netherlands.

The purposes of the present study were to explore the surgical possibilities for replacement of the medial tibial plateau by a metallic implant in a large animal model and to examine the implications for the opposing cartilage. In six goats, the medial tibial plateau of the right knee was replaced by a cobalt-chromium implant, using polymethylmethacrylate bone cement for fixation. The unoperated left knee served as a control. At 26 weeks after surgery, the animals were killed, and the joints evaluated macroscopically. Cartilage quality was analyzed macroscopically and histologically. Glycosaminoglycan content, synthesis, and release were measured in tissue and medium. All animals were able to move and load the knees without any limitations. Macroscopic articular evaluation scores showed worsening 26 weeks after inserting the implant (p < 0.05). Macroscopic and histologic scores showed more cartilage degeneration of the opposing medial femoral condyle in the experimental knee compared to the control knee (p < 0.05). Higher glycosaminoglycan synthesis was measured at the medial femoral condyle cartilage in the experimental knees (p < 0.05). This study shows that the medial tibial plateau can be successfully replaced by a cobalt-chromium implant in a large animal model. However, considerable femoral cartilage degeneration of the medial femoral condyle was induced, suggesting that care must be taken introducing hemiarthroplasty devices in a human clinical setting for the treatment of postmeniscectomy cartilage degeneration of the medial tibial plateau.
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http://dx.doi.org/10.1002/jor.21021DOI Listing
April 2010

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in vitro redifferentiation.

Am J Sports Med 2009 Nov 21;37 Suppl 1:97S-104S. Epub 2009 Oct 21.

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones.

Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).

Study Design: Controlled laboratory study.

Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).

Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.

Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

Clinical Relevance: Appreciation of the zonal differences can lead to important advances in cartilage tissue engineering. Findings support the use of hydrogels such as alginate for engineering zonal cartilage constructs.
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http://dx.doi.org/10.1177/0363546509350978DOI Listing
November 2009

Cartilage damage caused by metal implants applied for the treatment of established localized cartilage defects in a rabbit model.

J Orthop Res 2009 Jan;27(1):84-90

Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands.

The purpose of the current study was to investigate the feasibility of the application of defect-size femoral implants in a rabbit model of established cartilage defects and compare this treatment to microfracturing. In 31 New Zealand White rabbits, a medial femoral condyle defect was created in each knee. After 4 weeks, 3 animals were killed for defect baseline values. In the other 28 rabbits, knees were sham-operated, treated with microfracturing, or treated by placing an oxidized zirconium (OxZr) or cobalt-chromium (CoCr) implant (theta articulating surface 3.5 mm; fixating pin of 9.1 mm length). These animals were sacrificed 4 weeks after treatment. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry, and cartilage repair was scored microscopically. Cartilage quality was analyzed macroscopically and microscopically. Bone-implant contact was 63.2% +/- 3.2% for CoCr and 62.5% +/- 3.2% for OxZr. Cartilage defects did not show complete healing, nor during subsequent sham-surgery or microfracturing. For all treatments, considerable cartilage damage in the articulating medial tibia, and degeneration of lateral tibial and femoral cartilage was observed (p < 0.05). Both CoCr and OxZr implant-treated defects showed an increase of cartilage degeneration compared to microfracturing and sham-operated defects (p < 0.05). Although only a single short-term follow-up period was investigated in this study, caution is warranted using small metal implants as a treatment for established localized cartilage defects because, even after 4 weeks in this model, the metal implants caused considerable degeneration of the articulating surface.
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http://dx.doi.org/10.1002/jor.20709DOI Listing
January 2009

Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms.

Am J Physiol Heart Circ Physiol 2002 Oct;283(4):H1515-22

Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit University Medical Center, Amsterdam 1081 BT, The Netherlands.

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.
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http://dx.doi.org/10.1152/ajpheart.00113.2002DOI Listing
October 2002

Coronary end-to-side sleeve anastomosis using adhesive in off-pump bypass grafting in the pig.

Ann Thorac Surg 2002 May;73(5):1451-6

Heart Lung Center Utrecht, University Medical Center Utrecht, The Netherlands.

Background: In the exploration of facilitated coronary anastomosis strategies, we assessed a new octylcyanoacrylate adhesive in combination with a modified end-to-side sleeve anastomosis in off-pump bypass grafting in the pig.

Methods: Sleeve-adhesive anastomoses (n = 20) were evaluated intraoperatively, at 3 days (n = 4), and at 5 weeks (n = 16) in an off-pump, low (< or = 15 mL/min; n = 10) and high flow (approximately 60 mL/min; n = 10) porcine bypass model. All anastomoses were examined by flow measurement, angiography, and histology.

Results: Anastomosis construction took 8.5 minutes (6.7 to 10.2 minutes; median [15th to 85th percentile]). At 5 weeks, all anastomoses were fully patent (FitzGibbon grade A). The adhesive did not cause impaired vessel wall healing, but was surrounded by a focal acute and limited chronic (foreign body giant cells occasionally seen) inflammatory reaction at the adventitial application site.

Conclusions: Octyl-cyanoacrylate tissue adhesive combined with end-to-side internal mammary to coronary artery sleeve anastomosis construction proved to be feasible, even in low bypass graft flow conditions (< or = 15 mL/min; prothrombotic milieu) in the pig and deserves interest in exploration of facilitated anastomosis strategies in coronary artery bypass grafting.
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http://dx.doi.org/10.1016/s0003-4975(02)03423-9DOI Listing
May 2002