Enhanced microfracture using acellular scaffolds improves results after treatment of symptomatic focal grade III/IV knee cartilage lesions but current clinical evidence does not allow unequivocal recommendation.

PURPOSE: To systematically analyse post-operative outcomes following enhanced microfracture procedures in focal cartilage injuries of the knee. METHODS: Database searches were conducted in PubMed, EMBASE and Cochrane Library databases up to 30 November 2018, for clinical studies in humans that assessed surgical outcomes of enhanced microfracture procedures in focal cartilage injuries of the knee. The clinical, functional and imaging outcomes were assessed and summarized. The MINORS scale was used to assess the methodological quality of the studies included. RESULTS: Ten studies were included comprising a total of 331 patients (mean age of 37.0 ± 5.5 years, body mass 25.2 ± 1.7 kg m2, 56% male and 42% left knee), 278 femoral condyle chondral defects (147 medial, 35 lateral and 78 undefined) and 43 chondral defects distributed by the tibial plateau, patella and femoral trochlea. The chondral defects were mostly Outerbridge grade III or IV and the mean defect size was 3.2 ± 0.6 cm2. Studies consistently demonstrated significant improvement in the patient-reported outcome measures from baseline to final follow-up. Overall, imaging outcomes showed inconsistent results. Treatment-related adverse events were poorly reported. CONCLUSION: Enhanced microfracture techniques significantly result in improved patient-reported outcome measures over the MCID, but provide inconsistent imaging results. Current clinical evidence does not allow for unequivocal recommendation of enhanced microfracture to treat symptomatic focal grade III/IV knee cartilage lesions. LEVEL OF EVIDENCE: IV.

Mussel-Inspired Catechol Functionalisation as a Strategy to Enhance Biomaterial Adhesion: A Systematic Review.

Biomaterials have long been explored in regenerative medicine strategies for the repair or replacement of damaged organs and tissues, due to their biocompatibility, versatile physicochemical properties and tuneable mechanical cues capable of matching those of native tissues. However, poor adhesion under wet conditions (such as those found in tissues) has thus far limited their wider application. Indeed, despite its favourable physicochemical properties, facile gelation and biocompatibility, gellan gum (GG)-based hydrogels lack the tissue adhesiveness required for effective clinical use. Aiming at assessing whether substitution of GG by dopamine (DA) could be a suitable approach to overcome this problem, database searches were conducted on PubMed® and Embase® up to 2 March 2021, for studies using biomaterials covalently modified with a catechol-containing substituent conferring improved adhesion properties. In this regard, a total of 47 reports (out of 700 manuscripts, ~6.7%) were found to comply with the search/selection criteria, the majority of which (34/47, ~72%) were describing the modification of natural polymers, such as chitosan (11/47, ~23%) and hyaluronic acid (6/47, ~13%); conjugation of dopamine (as catechol "donor") via carbodiimide coupling chemistry was also predominant. Importantly, modification with DA did not impact the biocompatibility and mechanical properties of the biomaterials and resulting hydrogels. Overall, there is ample evidence in the literature that the bioinspired substitution of polymers of natural and synthetic origin by DA or other catechol moieties greatly improves adhesion to biological tissues (and other inorganic surfaces).

In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

Vascular disruption following spinal cord injury (SCI) decisively contributes to the poor functional recovery prognosis facing patients with the condition. Using a previously developed gellan gum hydrogel to which the adhesion motif GRGDS was grafted (GG-GRGDS), this work aimed to understand the ability of adipose-derived stem cells (ASCs) to impact vascular organization of human umbilical vein endothelial cells (HUVECs), and how this in turn affects neurite outgrowth of dorsal root ganglia (DRG) explants. Our data shows that culturing these cells together lead to a synergistic effect as showed by increased stimulation of neuritogenesis on DRG. Importantly, HUVECs were only able to assemble into vascular-like structures when cultured in the presence of ASCs, which shows the capacity of these cells in reorganizing the vascular milieu. Analysis of selected neuroregulatory molecules showed that the co-culture upregulated the secretion of several neurotrophic factors. On the other hand, ASCs, and ASCs + HUVECs presented a similar profile regarding the presence of angiotrophic molecules herein analyzed. Finally, the implantation of GG-GRGDS hydrogels encapsulating ASCs in the chick chorioallantoic membrane (CAM) lead to increases in vascular recruitment toward the hydrogels in comparison to GG-GRGDS alone. This indicates that the combination of ASCs with GG-GRGDS hydrogels could promote re-vascularization in trauma-related injuries in the central nervous system and thus control disease progression and induce functional recovery.

Synthesis and biological evaluation of a bioinspired, tissue-adhesive gellan gum-based hydrogel designed for minimally invasive delivery and retention of chondrogenic cells.

A dopamine-modified, bioinspired gellan gum hydrogel (STM-148B) with improved physicochemical and biological characteristics, suitable for minimally invasive cell delivery and retention in the context of cartilage repair, is herein presented. STM-148B's putative game-changing design characteristics include a highly biocompatible, animal-free and chemically defined composition, reproducibility of manufacture and ease of formulation. STM-148B undergoes rapid ionic crossinking by physiologically relevant mono and divalent cations to form stable 3D hydrogels that possess excellent tissue adhesiveness, such that additional fixation aids are rendered superfluous. STM-148B hydrogels maintain viability of mammalian cells and further promote up-regulation of the expression of healthy chondrogenic extracellular matrix markers upon stimulation. STM-148B is currently undergoing pre-clinical safety and efficacy assessment as a medical device for cell delivery and retention focussing on regeneration of hyaline-like cartilage and may represent a valuable addition to the armamentarium of tissue-engineering therapies for treatment of focal cartilage lesions.

Supercritical CO2 technology: The next standard sterilization technique?

Sterilization of implantable medical devices is of most importance to avoid surgery related complications such as infection and rejection. Advances in biotechnology fields, such as tissue engineering, have led to the development of more sophisticated and complex biomedical devices that are often composed of natural biomaterials. This complexity poses a challenge to current sterilization techniques which frequently damage materials upon sterilization. The need for an effective alternative has driven research on supercritical carbon dioxide (scCO2) technology. This technology is characterized by using low temperatures and for being inert and non-toxic. The herein presented paper reviews the most relevant studies over the last 15 years which cover the use of scCO2 for sterilization and in which effective terminal sterilization is reported. The major topics discussed here are: microorganisms effectively sterilized by scCO2, inactivation mechanisms, operating parameters, materials sterilized by scCO2 and major requirements for validation of such technique according to medical devices' standards.

Hydrogel-based magnetoelectric microenvironments for tissue stimulation.

The development of strategies to mimic the natural environment of tissues with engineered scaffolds remains one of the biggest challenges of tissue engineering. Hydrogels appear as suitable materials for this purpose due to their substantial water content, biocompatibility, and for being able to carry nanomaterials that introduce new functionalities to the hydrogel. The incorporation of magnetically responsive and, in particular, magnetoelectric materials into the hydrogel-based scaffolds are a promising approach for bone tissue engineering applications once it can promote not only tissue regeneration through magnetic to mechanic to electrical conversion/stimuli but also the external control of the scaffold by the application of magnetic fields. This work reports on a new CoFe2O4/ Methacrylated Gellan Gum (GGMA)/poly(vinylidene fluoride) (PVDF) hydrogel-based scaffold with 20 kPa Young's modulus and cell viability superior to 80%. The ≈ 1 µm thick PVDF/CoFe2O4 spheres added to GGMA gel (2 wt.%) exhibit 20 emu.g-1 magnetization saturation, 2.7 kOe magnetic coercivity and ß-phase contents ≈ 78%, leading to a piezoelectric response |d33| of ≈ 22 pC N-1 and a magnetoelectric response of Δ|d33| ≈ 6 pC N-1 at a DC magnetic field of 220 m T, as verified for the CoFe2O4/PVDF spheres with 20 wt.% filler content. Such characteristics allow novel tissue regeneration strategies approaches once CoFe2O4/GGMA/PVDF has a porous 3-D structure, biocompatibility, bioresorbability, and mechanical/electrical dynamic responses that can be triggered by an applied external magnetic field.

Efficient synthesis of 2-(trifluoromethyl)nicotinic acid derivatives from simple fluorinated precursors.

Novel routes to 2-trifluoromethyl-nicotinic acid derivatives have been developed involving synthesis of the pyridine ring. These pyridyl compounds serve as key intermediates in the manufacture of the recently discovered COMT inhibitor, 3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-2-(trifluoromethyl)pyridine 1-oxide.

The Role of Biomaterials as Angiogenic Modulators of Spinal Cord Injury: Mimetics of the Spinal Cord, Cell and Angiogenic Factor Delivery Agents.

Spinal cord injury (SCI) represents an extremely debilitating condition for which no efficacious treatment is available. One of the main contributors to the inhospitable environment found in SCI is the vascular disruption that happens at the moment of injury that compromises the blood-spinal cord barrier (BSCB) and triggers a cascade of events that includes infiltration of inflammatory cells, ischemia and intraparenchymal hemorrhage. Due to the unsatisfactory nature of revascularization following SCI, restoring vascular perfusion and the BSCB seems an interesting way of modulating the lesion environment into a regenerative phenotype, with a potential increase in functional recovery. Certain biomaterials possess interesting features to enhance SCI therapies, and in fact have been applied as angiogenic promoters in other pathologies. The present mini-review intends to highlight the contribution that biomaterials could make in the development of novel therapeutic solutions able to restore proper vascularization and the BSCB.

αvß3 and α5ß1 integrin-specific ligands: From tumor angiogenesis inhibitors to vascularization promoters in regenerative medicine?

Integrins are cell adhesion receptors predominantly important during normal and tumor angiogenesis. A sequence present on several extracellular matrix proteins composed of Arg-Gly-Asp (RGD) has attracted attention due to its role in cell adhesion mediated by integrins. The development of ligands that can bind to integrins involved in tumor angiogenesis and brake disease progression has resulted in new investigational drug entities reaching the clinical trial phase in humans. The use of integrin-specific ligands can be useful for the vascularization of regenerative medicine constructs, which remains a major limitation for translation into clinical practice. In order to enhance vascularization, immobilization of integrin-specific RGD peptidomimetics within constructs is a recommended approach, due to their high specificity and selectivity towards certain desired integrins. This review endeavours to address the potential of peptidomimetic-coated biomaterials as vascular network promoters for regenerative medicine purposes. Clinical studies involving molecules tracking active integrins in cancer angiogenesis and reasons for their failure are also addressed.

In vitro and in vivo performance of methacrylated gellan gum hydrogel formulations for cartilage repair.

Methacrylated gellan gum (GGMA) formulation is proposed as a second-generation hydrogel for controlled delivery of cartilage-forming cells into focal chondral lesions, allowing immediate in situ retention of cells and 3D filling of lesion volume, such approach deemed compatible with an arthroscopic procedure. Formulation optimization was carried out in vitro using chondrocytes and adipose mesenchymal stromal/stem cells (ASCs). A proof-of-concept in vivo study was conducted using a rabbit model with induced chondral lesions. Outcomes were compared with microfracture or non-treated control. Three grading scores were used to evaluate tissue repair after 8 weeks by macroscopic, histological and immunohistochemical analysis. Intense collagen type II and low collagen type I gene and protein expression were achieved in vitro by the ASC + GGMA formulation, in light with development of healthy chondral tissue. In vivo, this formulation promoted significantly superior de novo cartilage formation compared with the non-treated group. Maintenance of chondral height and integration with native tissue was further accomplished. The physicochemical properties of the proposed GGMA hydrogel exhibited highly favorable characteristics and biological performance both in vitro and in vivo, positioning itself as an attractive xeno-free biomaterial to be used with chondrogenic cells for a cost-effective treatment of focal chondral lesions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1987-1996, 2018.

Synthesis and biological evaluation of novel, peripherally selective chromanyl imidazolethione-based inhibitors of dopamine beta-hydroxylase.

A novel series of dopamine beta-hydroxylase (DBH) inhibitors was designed and synthesized incorporating modifications to the core structure of nepicastat 3, with the principal aim of discovering potent DBH inhibitors exerting minimal effects on dopamine (DA) and noradrenaline (NA) levels in the central nervous system. This study resulted in the identification of a potent, peripherally selective DBH inhibitor, (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione hydrochloride 54 (BIA 5-453). In experiments in mice and rats at T(max) (9 h after administration), 54 reduced NA levels in a dose-dependent manner in both the left atrium and the left ventricle, with the maximal inhibitory effect attained at a dose of 100 mg/kg. In contrast to that found in the heart, 54 failed to affect NA tissue levels in the brain. Compound 54 is thus presented as a candidate for clinical evaluation for the treatment of chronic heart failure and hypertension.

Synthesis and biological evaluation of a novel series of "ortho-nitrated" inhibitors of catechol-O-methyltransferase.

Novel regioisomeric "ortho-nitrated" catechols related to the catechol-O-methyltransferase (COMT) inhibitors BIA 3-202 3 and BIA 3-335 4 were synthesized and biologically evaluated. Changing the position of the nitro group from the "classical" meta- to the ortho-position relative to the side-chain substituent of the nitrocatechol pharmacophore exerted profound effects on selectivity and duration of COMT inhibition. Alkylaryl compounds 7a-d possessed shorter duration of action than their regioisomers, but 7b displayed reversed selectivity over 3 at 3 and 6 h, exhibiting preferential central inhibition. In the amino-substituted series, ortho-nitrated regioisomer 14k was less peripherally selective than 4 and short-acting, whereas decahydroquinoline 14g displayed an unprecedented combination of long-acting and selective peripheral inhibition. 7b could provide a useful tool to probe the pharmacological utility of short-acting, centrally selective COMT inhibitors in the treatment of depression in Parkinsonian patients, and 14g represents a promising candidate for clinical evaluation as an adjunct to L-Dopa therapy.

Synthesis, biological evaluation, and molecular modeling studies of a novel, peripherally selective inhibitor of catechol-O-methyltransferase.

A novel series of potent, peripherally selective, and long-acting inhibitors of catechol-O-methyltransferase (COMT) has been synthesized. The introduction and nature of heteroatom-containing substituents to the side-chain of the nitrocatechol pharmacophore was found to have a profound effect on both peripheral selectivity and duration of COMT inhibition in the mouse. This approach led to the discovery of 1-(3,4-dihydroxy-5-nitrophenyl)-3-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]-1-propanone hydrochloride 35 (BIA 3-335), which was found to possess a superior inhibitory profile in vivo over both the nonselective inhibitor tolcapone 1 and the peripherally selective but short-acting entacapone 2. In this model, 35 retained 75% inhibition of peripheral COMT at 6 h after oral administration, yet significantly, only a minor reduction of central (cerebral) COMT activity was observed. Molecular modeling techniques were applied to review the analysis of the ternary enzyme-inhibitor complex previously determined by X-ray crystallography and to provide a deeper understanding of the structure-activity relationships within this novel series. Furthermore, a computational approach was applied in an effort to elucidate the particular structural factors relevant to the poor blood-brain permeability of 35. In conclusion, the improved biological properties herein reported reveal 35 as a candidate for clinical studies as an adjunct to L-DOPA therapy for Parkinson's disease.

Synthesis of 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone and derivatives as potent and long-acting peripheral inhibitors of catechol-O-methyltransferase.

A homologous series of novel nitro-catechol structures (7a-7e) were synthesized and tested as inhibitors of the enzyme catechol-O-methyltransferase (COMT). Increasing chain length was found to have significant impact on both brain penetration and duration of COMT inhibition in the rat. Of this series, compound 7b (1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone) was found to exhibit the most potent and selective inhibition of peripheral COMT, with an inhibition profile more similar to entacapone 2 than tolcapone 1 (an equipotent peripheral and central inhibitor) but with much improved duration of action (7b, 70% inhibition and 2, 25% inhibition at 9 h after administration). The effects of structural modifications to 7b on COMT inhibitory profile were investigated, and it is concluded that the carbonyl group and preferably unsubstituted aromatic ring are essential features to maintain prolonged peripheral COMT inhibition. The introduction of the alpha-methylene group, the major structural difference between 7b and 1, would appear responsible for the observed enhancement in selectivity of peripheral COMT inhibition of 7b, which has more limited access to the brain than 1.

The chemistry of catechol-O-methyltransferase inhibitors.

Despite several drawbacks, levodopa (L-dopa) remains the gold standard drug for treatment of the symptoms of Parkinson's disease (PD). L-dopa is a pro-drug of dopamine and is used to elevate striatal levels of the neurotransmitter. One approach to provide a more continuous and sustained delivery of dopamine has targeted one of the principal enzymes responsible for metabolic deactivation of L-dopa, namely catechol-O-methyltransferase (COMT). The chapter will provide a perspective of the medicinal chemistry behind the discovery of several COMT inhibitors and discuss how certain physicochemical parameters, including aqueous solubility and lipophilicity, are thought to influence pharmacokinetic properties such as absorption, distribution, and bioavailability.

Discovery of a long-acting, peripherally selective inhibitor of catechol-O-methyltransferase.

Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a 1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide (BIA 9-1067)) was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation as an adjunct to L-Dopa therapy of Parkinson's disease.

A concise synthesis of the 3-O-beta-D- and 4'-O-beta-D-glucuronide conjugates of trans-resveratrol.

trans-Resveratrol ((E)-3,4',5-trihydroxystilbene, 1) is a phytoalexin produced naturally in plants and grape skins as a stress metabolite protecting against fungal attack. Widespread interest in this apparently structurally simple molecule and synthetic stilbene analogues has arisen in recent years due to the discovery of its antioxidant, antiinflammatory, and anti-carcinogenic activities, among others. Although O-conjugation with glucuronic acid in vivo is known to represent a significant metabolic pathway for polyphenolic compounds in general and 1 in particular, preclinical studies have been hampered by the lack of chemically pure, completely characterized reference standards of both regioisomeric 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 for adequate identification and quantification of these significant metabolites. The present work describes a concise, convergent synthesis of both 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 using a strategy based on a novel Heck coupling of iodoaryl-O-beta-d-glucuronate esters with appropriately substituted styrenes, such that highly pure multimilligram to gram quantities of both the 3-O-beta-d- and 4'-O-beta-d-glucuronide conjugates of 1 can now be conveniently synthesized.

Chemical synthesis and characterization of conjugates of a novel catechol-O-methyltransferase inhibitor.

BIA 3-202, 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenylethanone 3, is a novel, reversible, and tight-binding peripheral inhibitor of the enzyme catechol-O-methyltransferase (COMT), which is currently under clinical evaluation for the treatment of Parkinson's disease as an adjunct to current L-Dopa/peripheral decarboxylase inhibitor therapy. Chemically pure, well-characterized reference standards of conjugates of 3 were required for investigation of the routes of metabolism in several animal species (including humans) and for pharmacokinetic studies. The lack of suitable literature precedents for efficient, regioselective synthesis of nitrocatechol conjugate metabolites prompted us to develop efficient and highly selective chemical preparations of O-glucuronide and O-sulfate conjugates of 3 such that multigram quantities of excellent purity can now be conveniently synthesized. It is anticipated that these procedures could be applied to the synthesis of conjugates of other COMT inhibitors, also based on the 3-nitrocatechol pharmacophore.

Kinetics and crystal structure of catechol-o-methyltransferase complex with co-substrate and a novel inhibitor with potential therapeutic application.

Catechol-O-methyltransferase (COMT; E.C. 2.1.1.6) is a ubiquitous enzyme in nature that plays an important role in the metabolism of catechol neurotransmitters and xenobiotics. In particular, inactivation of drugs such as L-3,4-dihydroxyphenylalanine (L-DOPA) via O-methylation is of relevant pharmacological importance, because L-DOPA is currently the most effective drug used in the treatment of Parkinson's disease. This justified the interest in developing COMT inhibitors as potential adjuncts to L-DOPA therapy. The kinetics of inhibition by BIA 3-335 (1-[3,4-dihydroxy-5-nitrophenyl]-3-(N-3'-trifluormethylphenyl)-piperazine-1-propanone dihydrochloride) were characterized using recombinant rat soluble COMT. BIA 3-335 was found to act as a potent, reversible, tight-binding inhibitor of COMT with a K(i) of 6.0 +/- 1.6 nM and displaying a competitive inhibition toward the substrate binding site and uncompetitive inhibition toward the S-adenosyl-L-methionine (SAM) binding site. The 2.0-A resolution crystal structure of COMT in complex with its cosubstrate SAM and a novel inhibitor BIA 3-335 shows the atomic interactions between the important residues at the active site and the inhibitor. This is the first report of a three-dimensional structure determination of COMT complexed with a potent, reversible, and tight-binding inhibitor that is expected to have therapeutic applications.