Case report: Equine metacarpophalangeal joint partial and full thickness defects treated with allogenic equine synovial membrane mesenchymal stem/stromal cell combined with umbilical cord mesenchymal stem/stromal cell conditioned medium.

Here, we describe a case of a 5-year-old show-jumping stallion presented with severe lameness, swelling, and pain on palpation of the left metacarpophalangeal joint (MCj). Diagnostic imaging revealed full and partial-thickness articular defects over the lateral condyle of the third metacarpus (MC3) and the dorsolateral aspect of the first phalanx (P1). After the lesion's arthroscopic curettage, the patient was subjected to an innovative regenerative treatment consisting of two intra-articular injections of equine synovial membrane mesenchymal stem/stromal cells (eSM-MSCs) combined with umbilical cord mesenchymal stem/stromal cells conditioned medium (UC-MSC CM), 15 days apart. A 12-week rehabilitation program was accomplished, and lameness, pain, and joint effusion were remarkably reduced; however, magnetic resonance imaging (MRI) and computed tomography (CT) scan presented incomplete healing of the MC3's lesion, prompting a second round of treatment. Subsequently, the horse achieved clinical soundness and returned to a higher level of athletic performance, and imaging exams revealed the absence of lesions at P1, fulfillment of the osteochondral lesion, and cartilage-like tissue formation at MC3's lesion site. The positive outcomes suggest the effectiveness of this combination for treating full and partial cartilage defects in horses. Multipotent mesenchymal stem/stromal cells (MSCs) and their bioactive factors compose a novel therapeutic approach for tissue regeneration and organ function restoration with anti-inflammatory and pro-regenerative impact through paracrine mechanisms.

The application of Bonelike® Poro as a synthetic bone substitute for the management of critical-sized bone defects - A comparative approach to the autograft technique - A preliminary study.

The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.

Innovative tailor made dextran based membranes with excellent non-inflammatory response: In vivo assessment.

In this work, dextran based membranes with potential to be used as implantable devices in Tissue Engineering and Regenerative Medicine (TERM) were prepared by a straightforward strategy. Briefly, two polymers approved by the Food and Drug Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with methacrylate moieties, and subjected to photocrosslinking. Employing different weight ratios of each polymer in the formulations allowed to obtain transparent membranes with tunable physicochemical properties and low adverse host tissue response. Independently of the material, all formulations have shown to be thermally stable up to 300 °C whilst variations in the polymer ratio resulted in membranes with different glass transition temperatures (Tg) and flexibility. The swelling capacity ranged from 50% to 200%. On the other hand, in vitro hydrolytic degradation did not show to be material-dependent and all membranes maintained their structural integrity for more than 30 days, losing only 8-12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all membranes are capable of supporting cell adhesion and viability. The in vivo biocompatibility of membranes implanted subcutaneously in rats' dorsum indicate that M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as "slight-irritant" and "non-irritant", respectively. From the histological analysis performed on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis thereby excluding the presence of toxic degradation by-products deposited or accumulated in these tissues. In combination, these results suggest that the newly developed formulations hold great potential as engineered devices for biomedical applications, where the biological response of cells and tissues are greatly dependent on the physical and chemical cues provided by the substrate.

Dental pulp stem cells and Bonelike® for bone regeneration in ovine model.

Development of synthetic bone substitutes has arisen as a major research interest in the need to find an alternative to autologous bone grafts. Using an ovine model, the present pre-clinical study presents a synthetic bone graft (Bonelike®) in combination with a cellular system as an alternative for the regeneration of non-critical defects. The association of biomaterials and cell-based therapies is a promising strategy for bone tissue engineering. Mesenchymal stem cells (MSCs) from human dental pulp have demonstrated both in vitro and in vivo to interact with diverse biomaterial systems and promote mineral deposition, aiming at the reconstruction of osseous defects. Moreover, these cells can be found and isolated from many species. Non-critical bone defects were treated with Bonelike® with or without MSCs obtained from the human dental pulp. Results showed that Bonelike® and MSCs treated defects showed improved bone regeneration compared with the defects treated with Bonelike® alone. Also, it was observed that the biomaterial matrix was reabsorbed and gradually replaced by new bone during the healing process. We therefore propose this combination as an efficient binomial strategy that promotes bone growth and vascularization in non-critical bone defects.

Human umbilical cord blood plasma as an alternative to animal sera for mesenchymal stromal cells in vitro expansion - A multicomponent metabolomic analysis.

Mesenchymal Stromal cells (MSCs) have a potential role in cell-based therapies. Foetal bovine serum (FBS) is used to supplement the basal cell culture medium but presents several disadvantages and risks. Other alternatives have been studied, including human umbilical cord blood plasma (hUCBP), aiming at the development of xeno-free culturing protocols. A comparative characterization of multicomponent metabolic composition of hUCBP and commercial FBS based on Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical analysis was performed. The analysis of 1H-NMR spectra revealed both similarities and differences between the two proposed supplements. Similar metabolites (amino acids, glucose, lipids and nucleotides) were found in the hUCBP and FBS NMR spectra. The results show that the major difference between the metabolic profiles of the two proposed supplements are due to the significantly higher levels of glucose and lower levels of lactate, glutamate, alanine and branched chain amino acids in hUCBP. Similar or slightly different levels of important proteinogenic amino acids, as well as of nucleotides, lipids were found in the hUCBP and FBS. In order to validate it's suitability for cell culture, umbilical cord-MSCs (UC-MSCs) and dental pulp stem cells (DPSCs) were expanded using hUCBP. In both hMSCs, in vitro culture with hUCBP supplementation presented similar to improved metabolic performances when compared to FBS. The two cell types tested expressed different optimum hUCBP percentage content. For DPSCs, the optimum hUCBP content was 6% and for UC-MSCs, 4%. Cultured hMSCs displayed no changes in senescence indicators, as well as maintained characteristic surface marker's expression. FBS substitution was associated with an increase in early apoptosis events, in a dose dependent manner, as well as to slight up- and down-regulation of targeted gene's expression. Tri-lineage differentiation capacity was also influenced by the substitution of FBS by hUCBP.

Effects of Human Mesenchymal Stem Cells Isolated from Wharton's Jelly of the Umbilical Cord and Conditioned Media on Skeletal Muscle Regeneration Using a Myectomy Model.

Skeletal muscle has good regenerative capacity, but the extent of muscle injury and the developed fibrosis might prevent complete regeneration. The in vivo application of human mesenchymal stem cells (HMSCs) of the umbilical cord and the conditioned media (CM) where the HMSCs were cultured and expanded, associated with different vehicles to induce muscle regeneration, was evaluated in a rat myectomy model. Two commercially available vehicles and a spherical hydrogel developed by our research group were used. The treated groups obtained interesting results in terms of muscle regeneration, both in the histological and in the functional assessments. A less evident scar tissue, demonstrated by collagen type I quantification, was present in the muscles treated with HMSCs or their CM. In terms of the histological evaluation performed by ISO 10993-6 scoring, it was observed that HMSCs apparently have a long-term negative effect, since the groups treated with CM presented better scores. CM could be considered an alternative to the in vivo transplantation of these cells, as it can benefit from the local tissue response to secreted molecules with similar results in terms of muscular regeneration. Searching for an optimal vehicle might be the key point in the future of skeletal muscle tissue engineering.

Promoting nerve regeneration in a neurotmesis rat model using poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly: in vitro and in vivo analysis.

In peripheral nerves MSCs can modulate Wallerian degeneration and the overall regenerative response by acting through paracrine mechanisms directly on regenerating axons or upon the nerve-supporting Schwann cells. In the present study, the effect of human MSCs from Wharton's jelly (HMSCs), differentiated into neuroglial-like cells associated to poly (DL-lactide-ε-caprolactone) membrane, on nerve regeneration, was evaluated in the neurotmesis injury rat sciatic nerve model. Results in vitro showed successful differentiation of HMSCs into neuroglial-like cells, characterized by expression of specific neuroglial markers confirmed by immunocytochemistry and by RT-PCR and qPCR targeting specific genes expressed. In vivo testing evaluated during the healing period of 20 weeks, showed no evident positive effect of HMSCs or neuroglial-like cell enrichment at the sciatic nerve repair site on most of the functional and nerve morphometric predictors of nerve regeneration although the nociception function was almost normal. EPT on the other hand, recovered significantly better after HMSCs enriched membrane employment, to values of residual functional impairment compared to other treated groups. When the neurotmesis injury can be surgically reconstructed with an end-to-end suture or by grafting, the addition of a PLC membrane associated with HMSCs seems to bring significant advantage, especially concerning the motor function recovery.