Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-β3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-β3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-β3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-β3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-β3 increased over time and exhibited the highest peak stress in stress-relaxation (σ(pk)=1.489±0.389 kPa) at day 21 among all groups. Taken together, these results demonstrate that hMSCs encapsulated in photocrosslinked CMC hydrogels supplemented with TGF-β3 are capable of elaborating functional extracellular matrix consistent with the NP phenotype. Such MSC-laden hydrogels may have application in NP replacement therapies.

Objective: This study examined the effects of reduced and elevated weight bearing on post-traumatic osteoarthritis (PTOA) development, locomotor joint kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). Design: Twenty-one adult rats were subjected to MMT surgery of the left hindlimb and then assigned to one of three groups: (1) regular (i.e., no intervention), (2) hindlimb immobilization, or (3) treadmill running. Sham surgery was performed in four additional rats. Voluntary wheel run time/distance was measured, and 3D hindlimb kinematics were quantified during treadmill locomotion using biplanar radiography. Rats were euthanized 8 weeks after MMT or sham surgery, and the microstructure of the tibial cartilage and subchondral bone was quantified using contrast enhanced micro-CT. Results: All three MMT groups showed signs of PTOA (full-thickness lesions and/or increased cartilage volume) compared to the sham group, however the regular and treadmill-running groups had greater osteophyte formation than the immobilization group. For the immobilization group, increased volume was only observed in the anterior region of the cartilage. The treadmill-running group demonstrated a greater knee varus angle at mid-stance than the sham group, while the immobilization group demonstrated greater reduction in voluntary running than all the other groups at 2 weeks post-surgery. Conclusions: Elevated weight-bearing via treadmill running at a slow/moderate speed did not accelerate PTOA in MMT rats when compared to regular weight-bearing. Reduced weight-bearing via immobilization may attenuate overall PTOA but still resulted in regional cartilage degeneration. Overall, there were minimal differences in hindlimb kinematics and voluntary running between MMT and sham rats.

cartilage loss results in pain, the main symptoms of OA patients. OA pain involves a complex integration of sensory, affective, and cognitive processes. The correlation between structural damage of the joint and pain is weak, the prevalence of knee pain in patients with radiographic knee OA ranges from 15%e81% in different studies. Still, gradual increase of pain seems to be connected with OA progression and therefore pain is discussed as an indicator of disease activity. Recent MRI studies further revealed a positive relationship between inflammatory changes in the joint and pain raising the intriguing question of which inflammatory mechanisms and mediators might be involved in pain generation. Nociceptors are located throughout the joint in tissues peripheral to cartilage but not in cartilage itself. Increased nociceptive inputs from the joints cause complex changes in the central nervous system. In the state of central sensitization, nociceptive neurons at different levels of the neuraxis are hyperexcitable, and hence, the nociceptive processing is amplified. Peripheral nociceptors are important targets in analgesic therapy due to the fact that many pathological conditions such as inflammation induce receptor excitation and sensitization. Numerous ion channels and receptors take part in nociception and might function as targets in pain therapy. Therefore, we analyzed electrophysiological properties of neuronal cells under the influence of conditioned medium derived from chondrocytes and synoviocytes from OA patients. Methods: Neuroblastoma cells (MHH) were grown in the presence of conditioned medium (CM) derived from chondrocytes (CM-c) and synoviocytes (CM-s) isolated from OA patients. After 5 days of treatment MHH cells were tested for their electrophysiological behavior by the patch clamp technique. Expression of ion channels including the peripheral Na þ channels Nav1.6e1.9 and the K þ channels KCNQ2 and KCNQ3 respectively was assessed by qPCR. Both types of CM from all five patients were tested for the content of thirteen cytokines including MCP1, Il-6 and IL-8 via the human inflammation panel (Legendplex). Results: Whole cell Na þ current measurements revealed, in four out of five samples, a reduction of the maximal conductance (g max ) of MHH cells under CM-c, CM-s reduced g max in three out of five cases. The steady state activation in all cases was shifted to a more positive membrane potential (E m ), the inactivation and recovery kinetics were unchanged. For the K þ current a reduction in I max was observed in four out of five samples under both treatment conditions (CM-c and CM-s), the steady state activation was unchanged. Expression studies showed changes of voltage dependent Na þ channels with the most prominent effect for Nav1.7, the expression of the two K þ channels KCNQ2, 3 was down regulated. Results from the inflammation panel showed IL-6, IL-8 and MCP1 to be present in the CM-c and CM-s. Conclusions: Treatment with conditioned medium changes the electrophysiological behavior of neuroblasto...