The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.

Oral delivery of potent peptide drugs provides key formulation challenges in the pharmaceutical industry: stability, solubility, and permeability. Intestinal permeation enhancers (PEs) can overcome the low oral bioavailability by improving the drug permeability. Conventional in vitro and ex vivo models for assessing PEs fail to predict efficacy in vivo. Here, we compared Caco-2 cells cultured in the conventional static Transwell model to a commercially available continuous flow microfluidic Gut-on-a-Chip model. We determined baseline permeability of FITC-Dextan 3 kDa (FD3) in Transwell (5.3 ± 0.8 × 10-8 cm/s) vs Chip (3.2 ± 1.8 × 10-7 cm/s). We screened the concentration impact of two established PEs sodium caprate and sucrose monolaurate and indicated a requirement for higher enhancer concentration in the Chip model to elicit equivalent efficacy e.g., 10 mM sodium caprate in Transwells vs 25 mM in Chips. Fasted and fed state simulated intestinal fluids (FaSSIF/FeSSIF) were introduced into the Chip and increased basal FD3 permeability by 3-fold and 20-fold, respectively, compared to 4-fold and 4000-fold in Transwells. We assessed the utility of this model to peptides (Insulin and Octreotide) with PEs and observed much more modest permeability enhancement in the Chip model in line with observations in ex vivo and in vivo preclinical models. These data indicate that microfluidic Chip models are well suited to bridge the gap between conventional in vitro and in vivo models.

Formulation, Characterisation and Evaluation of the Antihypertensive Peptides, Isoleucine-Proline-Proline and Leucine-Lysine-Proline in Chitosan Nanoparticles Coated with Zein for Oral Drug Delivery.

Isoleucine-Proline-Proline (IPP) and Leucine-Lysine-Proline (LKP) are food-derived tripeptides whose antihypertensive functions have been demonstrated in hypertensive rat models. However, peptides display low oral bioavailability due to poor intestinal epithelial permeability and instability. IPP and LKP were formulated into nanoparticles (NP) using chitosan (CL113) via ionotropic gelation and then coated with zein. Following addition of zein, a high encapsulation efficiency (EE) (>80%) was obtained for the NP. In simulated gastric fluid (SGF), 20% cumulative release of the peptides was achieved after 2 h, whereas in simulated intestinal fluid (SIF), ~90% cumulative release was observed after 6 h. Higher colloidal stability (39−41 mV) was observed for the coated NP compared to uncoated ones (30−35 mV). In vitro cytotoxicity studies showed no reduction in cellular viability of human intestinal epithelial Caco-2 and HepG2 liver cells upon exposure to NP and NP components. Administration of NP encapsulating IPP and LKP by oral gavage to spontaneously hypertensive rats (SHR) attenuated systolic blood pressure (SBP) for 8 h. This suggests that the NP provide appropriate release to achieve prolonged hypotensive effects in vivo. In conclusion, chitosan-zein nanoparticles (CZ NP) have potential as oral delivery system for the encapsulation of IPP and LKP.

Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD.

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

Effect of different hydroxyapatite incorporation methods on the structural and biological properties of porous collagen scaffolds for bone repair.

Scaffolds which aim to provide an optimised environment to regenerate bone tissue require a balance between mechanical properties and architecture known to be conducive to enable tissue regeneration, such as a high porosity and a suitable pore size. Using freeze-dried collagen-based scaffolds as an analogue of native ECM, we sought to improve the mechanical properties by incorporating hydroxyapatite (HA) in different ways while maintaining a pore architecture sufficient to allow cell infiltration, vascularisation and effective bone regeneration. Specifically we sought to elucidate the effect of different hydroxyapatite incorporation methods on the mechanical, morphological, and cellular response of the resultant collagen-HA scaffolds. The results demonstrated that incorporating either micron-sized (CHA scaffolds) or nano-sized HA particles (CnHA scaffolds) prior to freeze-drying resulted in moderate increases in stiffness (2.2-fold and 6.2-fold, respectively, vs. collagen-glycosaminoglycan scaffolds, P < 0.05, a scaffold known to support osteogenesis), while enabling good cell attachment, and moderate mesenchymal stem cell (MSC)-mediated calcium production after 28 days' culture (2.1-fold, P < 0.05, and 1.3-fold, respectively, vs. CG scaffolds). However, coating of collagen scaffolds with a hydroxyapatite precipitate after freeze-drying (CpHA scaffolds) has been shown to be a highly effective method to increase the compressive modulus (26-fold vs. CG controls, P < 0.001) of scaffolds while maintaining a high porosity (~ 98%). The coating of the ligand-dense collagen structure results in a lower cell attachment level (P < 0.05), although it supported greater cell-mediated calcium production (P < 0.0001) compared with other scaffold variants after 28 days' culture. The comparatively good mechanical properties of these high porosity scaffolds is obtained partially through highly crosslinking the scaffolds with both a physical (DHT) and chemical (EDAC) crosslinking treatment. Control of scaffold microstructure was examined via alterations in freezing temperature. It was found that the addition of HA prior to freeze-drying generally reduced the pore size and so the CpHA scaffold fabrication method offered increased control over the resulting scaffolds microstructure. These findings will help guide future design considerations for composite biomaterials and demonstrate that the method of HA incorporation can have profound effects on the resulting scaffold structural and biological response.

Novel microhydroxyapatite particles in a collagen scaffold: a bioactive bone void filler?

BACKGROUND: Treatment of segmental bone loss remains a major challenge in orthopaedic surgery. Traditional techniques (eg, autograft) and newer techniques (eg, recombinant human bone morphogenetic protein-2 [rhBMP-2]) have well-established performance limitations and safety concerns respectively. Consequently there is an unmet need for osteoinductive bone graft substitutes that may eliminate or reduce the use of rhBMP-2. QUESTIONS/PURPOSES: Using an established rabbit radius osteotomy defect model with positive (autogenous bone graft) and negative (empty sham) control groups, we asked: (1) whether a collagen-glycosaminoglycan scaffold alone can heal the defect, (2) whether the addition of hydroxyapatite particles to the collagen scaffold promote faster healing, and (3) whether the collagen-glycosaminoglycan and collagen-hydroxyapatite scaffolds are able to promote faster healing (by carrying a low dose rhBMP-2). METHODS: A 15-mm transosseous radius defect in 4-month-old skeletally mature New Zealand White rabbits were treated with either collagen-hydroxyapatite or collagen-glycosaminoglycan scaffolds with and without rhBMP-2. Autogenous bone graft served as a positive control. Time-series radiographs at four intervals and postmortem micro-CT and histological analysis at 16 weeks were performed. Qualitative histological analysis of postmortem explants, and qualitative and volumetric 3-D analysis of standard radiographs and micro-CT scans enabled direct comparison of healing between test groups. RESULTS: Six weeks after implantation the collagen-glycosaminoglycan group had callus occupying greater than ½ the defect, whereas the sham (empty) control defect was still empty and the autogenous bone graft defect was completely filled with unremodeled bone. At 6 weeks, the collagen-hydroxyapatite scaffold groups showed greater defect filling with dense callus compared with the collagen-glycosaminoglycan controls. At 16 weeks, the autogenous bone graft groups showed evidence of early-stage medullary canal formation beginning at the proximal and distal defect borders. The collagen-glycosaminoglycan and collagen-glycosaminoglycan-rhBMP-2 groups had nearly complete medullary canal formation and anatomic healing at 16 weeks. However, collagen-hydroxyapatite-rhBMP-2 scaffolds showed the best levels of healing, exhibiting a dense callus which completely filled the defect. CONCLUSIONS: The collagen-hydroxyapatite scaffold showed comparable healing to the current gold standard of autogenous bone graft. It also performed comparably to collagen-glycosaminoglycan-rhBMP-2, a representative commercial device in current clinical use, but without the cost and safety concerns. CLINICAL RELEVANCE: The collagen-glycosaminoglycan scaffold may be suitable for a low load-bearing defect. The collagen-hydroxyapatite scaffold may be suitable for a load-bearing defect. The rhBMP-2 containing collagen-glycosaminoglycan and collagen-hydroxyapatite scaffolds may be suitable for established nonunion defects.

Validation of a Caco-2 microfluidic Chip model for predicting intestinal absorption of BCS Class I-IV drugs.

Oral delivery is considered the most patient preferred route of drug administration, however, the drug must be sufficiently soluble and permeable to successfully formulate an oral formulation. There have been advancements in the development of more predictive solubility and dissolution tools, but the tools that has been developed for permeability assays have not been validated as extensively as the gold-standard Caco-2 Transwell assay. Here, we evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (Papp), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (fa) in both culture conditions. Permeability differences were observed across the models as demonstrated by antipyrine (Transwell Papp: 38.5 ± 6.1 × 10-8 cm/s vs Chip Papp: 32.9 ± 11.3 × 10-8 cm/s) and nadolol (Transwell Papp: 0.6 ± 0.1 × 10-7 cm/s vs Chip Papp: 3 ± 1.2 × 10-7 cm/s). The in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r2 = 0.59-0.83) was similar to the Chip model (r2 = 0.41-0.79), highlighting similar levels of predictivity. Comparing to historical data, our Chip Papp data was more closely aligned to native tissues assessed in Ussing chambers. This is the first study to comprehensively validate a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption to further reduce our reliance on animal models.

Long-term daily oral administration of intestinal permeation enhancers is safe and effective in mice.

Although protein drugs are powerful biologic therapeutics, they cannot be delivered orally because their large size and hydrophilicity limit their absorption across the intestinal epithelium. One potential solution is the incorporation of permeation enhancers into oral protein formulations; however, few have advanced clinically due to toxicity concerns surrounding chronic use. To better understand these concerns, we conducted a 30-day longitudinal study of daily oral permeation enhancer use in mice and resultant effects on intestinal health. Specifically, we investigated three permeation enhancers: sodium caprate (C10), an industry standard, as well as 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC). Over 30 days of treatment, all mice gained weight, and none required removal from the study due to poor health. Furthermore, intestinal permeability did not increase following chronic use. We also quantified the gene expression of four tight junction proteins (claudin 2, claudin 3, ZO-1, and JAM-A). Significant differences in gene expression between untreated and permeation enhancer-treated mice were found, but these varied between treatment groups, with most differences resolving after a 1-week washout period. Immunofluorescence microscopy revealed no observable differences in protein localization or villus architecture between treated and untreated mice. Overall, PPZ and SDC performed comparably to C10, one of the most clinically advanced enhancers, and results suggest that the chronic use of some permeation enhancers may be therapeutically viable from a safety standpoint.

Highly Porous Type II Collagen-Containing Scaffolds for Enhanced Cartilage Repair with Reduced Hypertrophic Cartilage Formation.

The ability to regenerate damaged cartilage capable of long-term performance in an active joint remains an unmet clinical challenge in regenerative medicine. Biomimetic scaffold biomaterials have shown some potential to direct effective cartilage-like formation and repair, albeit with limited clinical translation. In this context, type II collagen (CII)-containing scaffolds have been recently developed by our research group and have demonstrated significant chondrogenic capacity using murine cells. However, the ability of these CII-containing scaffolds to support improved longer-lasting cartilage repair with reduced calcified cartilage formation still needs to be assessed in order to elucidate their potential therapeutic benefit to patients. To this end, CII-containing scaffolds in presence or absence of hyaluronic acid (HyA) within a type I collagen (CI) network were manufactured and cultured with human mesenchymal stem cells (MSCs) in vitro under chondrogenic conditions for 28 days. Consistent with our previous study in rat cells, the results revealed enhanced cartilage-like formation in the biomimetic scaffolds. In addition, while the variable chondrogenic abilities of human MSCs isolated from different donors were highlighted, protein expression analysis illustrated consistent responses in terms of the deposition of key cartilage extracellular matrix (ECM) components. Specifically, CI/II-HyA scaffolds directed the greatest cell-mediated synthesis and accumulation in the matrices of type II collagen (a principal cartilage ECM component), and reduced deposition of type X collagen (a key protein associated with hypertrophic cartilage formation). Taken together, these results provide further evidence of the capability of these CI/II-HyA scaffolds to direct enhanced and longer-lasting cartilage repair in patients with reduced hypertrophic cartilage formation.

Profiling of mature-stage human breast milk cells identifies six unique lactocyte subpopulations.

Breast milk is chock-full of nutrients, immunological factors, and cells that aid infant development. Maternal cells are the least studied breast milk component, and their unique properties are difficult to identify using traditional techniques. Here, we characterized the cells in mature-stage breast milk from healthy donors at the protein, gene, and transcriptome levels. Holistic analysis of flow cytometry, quantitative polymerase chain reaction, and single-cell RNA sequencing data identified the predominant cell population as epithelial with smaller populations of macrophages and T cells. Two percent of epithelial cells expressed four stem cell markers: SOX2, TRA-1-60, NANOG, and SSEA4. Furthermore, milk contained six distinct epithelial lactocyte subpopulations, including three previously unidentified subpopulations programmed toward mucosal defense and intestinal development. Pseudotime analysis delineated the differentiation pathways of epithelial progenitors. Together, these data define healthy human maternal breast milk cells and provide a basis for their application in maternal and infant medicine.

A highly porous type II collagen containing scaffold for the treatment of cartilage defects enhances MSC chondrogenesis and early cartilaginous matrix deposition.

A major challenge in cartilage tissue engineering (TE) is the development of instructive and biomimetic scaffolds capable of driving effective mesenchymal stem cell (MSC) chondrogenic differentiation and robust de novo matrix formation. Type I collagen-based scaffolds are one of the most commonly selected materials given collagen's intrinsic ability to act as an instructive and active biomaterial. However, the chondrogenic potential of these scaffolds does not offer significant improvement over traditional treatments. We propose that taking a biomimetic approach to scaffold development might lead to an improved outcome for enhanced cartilage repair. Therefore, this study aimed to develop innovative type II collagen (CII)-containing scaffolds for enhanced cartilage repair, by incorporating CII and/or hyaluronic acid (HyA) into a type I collagen (CI) framework. Moreover, focus was placed on understanding the potential synergistic effects played by CII in combination with HyA, in terms of MSC chondrogenesis and cartilage-like formation, when both molecules are incorporated into scaffold biomaterials. The newly developed CII-containing scaffold exhibited a highly porous interconnected structure with 99% porosity and similar mechanical properties to previously optimised collagen-based scaffolds. Although all scaffold variants sustained early cartilaginous matrix deposition, the CII-containing scaffolds in the presence of HyA performed best, offering enhanced deposition and distribution of sulphated glycosaminoglycans (sGAG) in vitro by day 28. Taken together, the combination of CII and HyA resulted in the development of a biomimetic scaffold with improved chondrogenic benefits. These simple "off-the-shelf" implants hold great promise to direct enhanced tissue regeneration for the treatment of focal cartilage defects.

Intestinal permeation enhancers enable oral delivery of macromolecules up to 70 kDa in size.

The decades-long effort to deliver peptide drugs orally has resulted in several clinically successful formulations. These formulations are enabled by the inclusion of permeation enhancers that facilitate the intestinal absorption of peptides. Thus far, these oral peptide drugs have been limited to peptides less than 5 kDa, and it is unclear whether there is an upper bound of protein size that can be delivered with permeation enhancers. In this work, we examined two permeation enhancers, 1-phenylpiperazine (PPZ) and sodium deoxycholate (SDC), for their ability to increase intestinal transport of a model macromolecule (FITC-Dextran) as a function of its size. Specifically, the permeability of dextrans with molecular weights of 4, 10, 40, and 70 kDa was assessed in an in vitro and in vivo model of the intestine. In Caco-2 monolayers, both PPZ and SDC significantly increased the permeability of only FD4 and FD10. However, in mice, PPZ and SDC behaved differently. While SDC improved the absorption of all tested sizes of dextrans, PPZ was effective only for FD4 and FD10. This work is the first report of PPZ as a permeation enhancer in vivo, and it highlights the ability of permeation enhancers to improve the absorption of macromolecules across a broad range of sizes relevant for protein drugs.

Oral delivery of peptide therapeutics in infants: Challenges and opportunities.

The vast majority of drugs are not designed or developed for pediatric and infant populations. Peptide drugs, which have become increasingly relevant in the past several decades, are no exception. Unfortunately, nearly all of the 60+ approved peptide drugs are formulated for injection, a particularly unfriendly mode of administration for infants. Although three peptide drugs were recently approved for oral formulations, this major advance in peptide drug delivery is available only for adults. In this review, we consider the current challenges and opportunities for the oral formulation of peptide therapeutics, specifically for infant populations. We describe the strategies that enable oral protein delivery and the potential impact of infant physiology on those strategies. We also detail the limited but encouraging progress towards 1) adapting conventional drug development and delivery approaches to infants and 2) designing novel infant-centric formulations. Together, these efforts underscore the feasibility of oral peptide delivery in infants and provide motivation to increase attention paid to this underserved area of drug delivery and formulation.

The enhanced intestinal permeability of infant mice enables oral protein and macromolecular absorption without delivery technology.

Oral delivery of macromolecular drugs is the most patient-preferred route of administration because it is painless and convenient. Over the past 30 years, significant attention has been paid to oral protein delivery in adults. Unfortunately, there is an outstanding need for similar efforts in infants, a patient population with distinct intestinal physiology and treatment needs. Here, we assess the intestinal permeability of neonatal and infant mice to determine the feasibility of orally delivering peptide and protein drugs without permeation enhancers or other assistance. Using the non-everted gut sac model, we found that macromolecular permeability depended on molecular size, mouse age, and intestinal tissue type using model dextrans. For example, the apparent permeability of 70 kDa FITC-Dextran (FD70) in infant small intestinal tissue was 2-5-fold higher than in adult tissue. As mice aged, the expression of barrier-forming and pore-forming tight junction proteins increased and decreased, respectively. The in vivo oral absorption of 4 kDa FITC-Dextran (FD4) and FD70 was significantly higher in younger mice, and there was a fourfold increase in oral absorption of the 80 kDa protein lactoferrin compared to adults. Oral gavage of insulin (5 IU/kg) reduced blood glucose levels in infants by >20% at 2 and 3 h but had no effect in adults. Oral insulin had 35% and <1% of the pharmacodynamic effect of a 1 IU/kg subcutaneous dose in infants and adults, as measured by area above the curve. These data indicate that the uniquely leaky nature of the infantile intestine may support the oral delivery of biologics without the need for traditional oral delivery technology.

Striving Towards the Perfect In Vitro Oral Drug Absorption Model.

Oral drug delivery systems have multiple goals, assessing and enabling intestinal absorption at efficacious doses being one of them. Here we highlight the in vitro advances in modeling drug absorption, which more faithfully reflect human intestinal physiology and reduce the reliance on animal models.

Growth Hormone Induces Colon DNA Damage Independent of IGF-1.

DNA damage occurs as a result of environmental insults and aging and, if unrepaired, may lead to chromosomal instability and tumorigenesis. Because GH suppresses ataxia-telangiectasia mutated kinase phosphorylation, decreases DNA repair, and increases DNA damage accumulation, we elucidated whether GH effects on DNA damage are mediated through induced IGF-1. In nontumorous human colon cells, GH, but not IGF-1, increased DNA damage. Stably disrupted IGF-1 receptor (IGF-1R) by lentivirus-expressing short hairpin RNA in vitro or treatment with the IGF-1R phosphorylation inhibitor picropodophyllotoxin (PPP) in vitro and in vivo led to markedly induced GH receptor (GHR) abundance, rendering cells more responsive to GH actions. Suppressing IGF-1R triggered DNA damage in both normal human colon cells and three-dimensional human intestinal organoids. DNA damage was further increased when cells with disrupted IGF-1R were treated with GH. Because GH induction of DNA damage accumulation appeared to be mediated not by IGF-1R but probably by more abundant GH receptor expression, we injected athymic mice with GH-secreting xenografts and then treated them with PPP. In these mice, high circulating GH levels were associated with increased colon DNA damage despite disrupted IGF-1R activity (P < 0.01), whereas GHR levels were also induced. Further confirming that GH effects on DNA damage are directly mediated by GHR signaling, GHR-/- mice injected with PPP did not show increased DNA damage, whereas wild-type mice with intact GHR exhibited increased colon DNA damage in the face of IGF-1 signaling suppression. The results indicate that GH directly induces DNA damage independent of IGF-1.

Development of a collagen calcium-phosphate scaffold as a novel bone graft substitute.

Previous investigations have shown that collagen shows excellent biological performance as a scaffold for tissue engineering. As a primary constituent of bone and cartilage, it demonstrates excellent cell adhesion and proliferation. However, in bone tissue engineering, it has insufficient mechanical properties for implantation in a load-bearing defect. The objective of this preliminary study was to investigate the possibility of developing a collagen/calcium-phosphate composite scaffold which would combine the biological performance and the high porosity of a collagen scaffold with the high mechanical stiffness of a calcium-phosphate scaffold. Collagen scaffolds were produced by a lyophilisation process from a collagen slurry. The scaffolds were soaked for different exposure times in solutions of 0.1 M, 0.5 M or 1.0 M NaNH4HPO4 followed by 0.1 M, 0.5 M or 1.0 M CaCl2. Mechanical tests of each scaffold were performed on a uniaxial testing system. Young's moduli were determined from stress-strain curves. The pore structure and porosity of the scaffolds were investigated using micro-computed tomography. A pure collagen scaffold served as a control. All scaffolds showed a significantly increased compressive stiffness relative to the pure collagen scaffolds. The exposure to the 0.5 M solutions showed significantly superior results compared to the other groups. Analysis of the pore structure indicated a decrease in the overall porosity of the composite scaffolds relative to the controls. Regarding mechanical stiffness and porosity, scaffolds after 1 hour exposure to the 0.5 M solutions showed the best properties for bone tissue engineering. Further work will involve producing a scaffold with a more homogeneous calcium phosphate distribution.

Sodium caprate enables the blood pressure-lowering effect of Ile-Pro-Pro and Leu-Lys-Pro in spontaneously hypertensive rats by indirectly overcoming PepT1 inhibition.

The tripeptides, Ile-Pro-Pro (IPP) and Leu-Lys-Pro (LKP), inhibit angiotensin-converting enzyme (ACE) resulting in lowered blood pressure. Our hypothesis was that the medium chain fatty acid permeation enhancer, sodium caprate (C10), may prevent the decrease in permeability of the tripeptides when PepT1 is inhibited by glycyl-sarcosine (Gly-Sar), a situation that may occur in the presence of food hydrolysates. Using Caco-2 monolayers and isolated rat jejunal tissue, the apparent permeability coefficients (Papp) of [3H]-IPP and [3H]-LKP were assessed in the presence of Gly-Sar with and without C10. Gly-Sar decreased the Papp of both tripeptides across monolayers and isolated jejunal tissue, but C10 restored it. C10 likely increased the paracellular permeability of the tripeptides, as indicated by immunofluorescence changes in tight junction proteins in Caco-2 monolayers accompanied by a concentration-dependent decrease in transepithelial electrical resistance (TEER). [3H]-IPP and [3H]-LKP were orally-gavaged to normal rats with Gly-Sar, C10, or with a mixture. Plasma levels of both peptides were reduced by Gly-Sar to less than half that of the levels detected in its absence, but were restored when C10 was co-administered. In spontaneously hypertensive rats (SHRs), unlabelled IPP and LKP lowered blood pressure when delivered either by i.v. or oral routes. Oral gavage of Gly-Sar reduced the hypotensive action of peptides in SHRs, but the effect was restored in the presence of C10. In conclusion, there was a reduction in the hypotensive effects of IPP and LKP in SHRs when intestinal PepT1 was inhibited by Gly-Sar, but C10 may circumvent this by enhancing paracellular permeability.

An endochondral ossification approach to early stage bone repair: Use of tissue-engineered hypertrophic cartilage constructs as primordial templates for weight-bearing bone repair.

Mimicking endochondral ossification to engineer constructs offers a novel solution to overcoming the problems associated with poor vascularisation in bone repair. This can be achieved by harnessing the angiogenic potency of hypertrophic cartilage. In this study, we demonstrate that tissue-engineered hypertrophically primed cartilage constructs can be developed from collagen-based scaffolds cultured with mesenchymal stem cells. These constructs were subsequently implanted into femoral defects in rats. It was evident that the constructs could support enhanced early stage healing at 4 weeks of these weight-bearing femoral bone defects compared to untreated defects. This study demonstrates the value of combining knowledge of development biology and tissue engineering in a developmental engineering inspired approach to tissue repair.

Enhanced Utilization of Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids Using Microengineered Chips.

BACKGROUND AND AIMS: Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium's role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell-derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli. METHODS: Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate-dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function. RESULTS: The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability. CONCLUSIONS: We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.