Healing properties of surface-coated polycaprolactone-co-lactide scaffolds: a pilot study in sheep.

The aim of this pilot study was to evaluate the bioactive, surface-coated polycaprolactone-co-lactide scaffolds as bone implants in a tibia critical size defect model. Polycaprolactone-co-lactide scaffolds were coated with collagen type I and chondroitin sulfate and 30 piled up polycaprolactone-co-lactide scaffolds were implanted into a 3 cm sheep tibia critical size defect for 3 or 12 months (n = 5 each). Bone healing was estimated by quantification of bone volume in the defects on computer tomography and microcomputer tomography scans, plain radiographs, biomechanical testing as well as by histological evaluations. New bone formation occurred at the proximal and distal ends of the tibia in both groups. The current pilot study revealed a mean new bone formation of 63% and 172% after 3 and 12 months, respectively. The bioactive, surface coated, highly porous three-dimensional polycaprolactone-co-lactide scaffold stack itself acted as a guide rail for new bone formation along and into the implant. These preliminary data are encouraging for future experiments with a larger group of animals.

Long-bone critical-size defects treated with tissue-engineered polycaprolactone-co-lactide scaffolds: a pilot study on rats.

The aim of this study was to evaluate the osteogenic potential of embroidered, tissue-engineered polycaprolactone-co-lactide (trade name: PCL) scaffolds for the reconstruction of large bone defects. Ten piled-up PCL scaffolds were implanted in femura with a critical size defect of immunodeficient nude rats for 12 weeks [n = 4, group 1: noncoated, group 2: collagen I (coll I), group 3: collagen I/chondroitin sulfate (coll I/CS), and group 4: collagen I/chondroitin sulfate/human mesenchymal stem cells (coll I/CS/hMSC)]. X-ray examination, computer tomography, and histological analyses of the explanted scaffold pads were performed. The quantification of the bone volume ratio showed a significantly higher rate of new bone formation at coll I/CS-coated scaffolds compared with the other groups. Histological investigations revealed that the defect reconstruction started from the peripheral bone ends and incorporated into the scaffold material. Additionally seeded hMSC on coll I/CS-coated scaffolds showed a higher matrix deposition inside the implant but no higher bone formation was observed. These data imply that the coll I/CS-coated PCL scaffolds have the highest potential for treating critical size defects. The scaffolds, being variable in size and structure, can be adapted to any bone defect.

Pharmacological modification of the epithelial permeability by benzalkonium chloride in UVA/Riboflavin corneal collagen cross-linking.

PURPOSE: To examine the biomechanical effect and the UVA-absorption of a riboflavin/UVA cross-linking method, which suggests leaving the epithelium intact and applying benzalkonium chloride (BAC) on rabbits' corneas. METHODS: In total, 32 eyes from 16 rabbits were divided into 4 groups. Group 1 was treated with intact epithelium and without BAC. In groups 2 and 3, the epithelium was left intact and a hypoosmolar solution of riboflavin that contained BAC 0.02% or 0.04% was used. Group 4 was treated according to the standard protocol with mechanical debridement of the epithelium. After the treatment of both eyes, the rabbits were euthanized to prepare the corneas in order for the determination of the riboflavin absorption coefficient and biomechanical properties. RESULTS: The absorption coefficients of groups 2, 3, and 4 were significantly increased compared to group 1. There were no significant differences between groups 2, 3, and 4. Stress-strain values and Young's modulus for groups 2, 3, and 4 were significantly increased compared to group 1. The stiffening effects did not differ within groups 2, 3, and 4. The resistance to enzymatic digestion was significantly increased in groups 2, 3, and 4 as compared to group 1. CONCLUSIONS: Treatment with BAC 0.02% induces sufficient epithelial permeability for the passage of riboflavin, which enables its stromal diffusion and results in increased corneal stiffening after cross-linking as compared to the standard protocol. Further safety studies will be required before clinical use.

Reduction of oral mucositis by palifermin (rHuKGF): dose-effect of rHuKGF.

PURPOSE: The aim of the present study was to determine the dose effect of palifermin (recombinant human keratinocyte growth factor, rHuKGF) for reduction of the response of oral mucosa to fractionated radiotherapy in a mouse model. MATERIAL AND METHODS: Ulceration (confluent mucositis) of mouse tongue epithelium was analysed as the clinically relevant endpoint. Palifermin at doses from 1 - 30 mg/kg was administered before the onset (day -3), at the end of the first (day +4) or the second week of irradiation (day +11) with 5 x 3 Gy/week. Each protocol was terminated by graded radiation test (top-up) doses. In a further experiment, optimally effective doses were given on days -3 and +4, or -3, +4 and +11. RESULTS: Single dose irradiation of mouse mucosa yielded an ED50 (dose inducing ulcer in 50% of the mice) of 10.7 +/- 1.0 Gy. With fractionated irradiation for 1 week an ED50 for test irradiation (day +7) of 5.1 +/- 1.9 Gy was observed. After 2 weeks (day +14), the ED50 was 7.3 +/- 1.9 Gy. Palifermin significantly increased the ED50 values in all protocols tested. Maximally effective doses for single injections were 15.0 mg/kg (day -3, +11) or 22.5 mg/kg (day +4), which yielded ED50 values of 12.1 +/- 1.3 Gy, 13.7 +/- 1.5 Gy and 14.4 +/- 1.3 Gy, respectively. Higher palifermin doses did not further increase the ED50. Repeated injections on days -3 and +4 did not increase the ED50 beyond the value obtained with injections on day +4 alone. An additional injection on day +11 increased the ED50 further to 15.1 +/- 0.1 Gy. CONCLUSIONS: A significant palifermin dose-effect was seen at doses below 15 mg/kg. However, a significant increase in oral mucosal radiation tolerance by palifermin over untreated control tissue was observed already with low doses of 1 mg/kg. This indicates that in clinical studies with palifermin, the dose of the growth factor may be of minor relevance over a wide dose range.

Reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): experimental studies in mice.

PURPOSE: To study the effect of recombinant human keratinocyte growth factor (rHuKGF or palifermin) on oral mucositis induced by radiochemotherapy in a mouse model. METHODS AND MATERIALS: Cis-diamminedichloroplatinum (cisplatin) and/or 5-fluorouracil were given before single dose irradiation, combined with palifermin before or after the treatment, or both. Daily fractionated irradiation for 2 weeks was followed by graded test doses. With additional chemotherapy in Week 1, palifermin was given before radiotherapy and at the end of the first week, or additionally at the end of Week 2. Radiochemotherapy in Week 2 was combined with palifermin at the end of Weeks 1 and 2, Weeks 1, 2, and 3, or additionally before radiotherapy. Ulceration of mouse tongue mucosa was analyzed as the endpoint. RESULTS: The dose associated with ulcer induction in 50% of the mice (ED(50)) for single-dose irradiation was 11.5 +/- 0.7 Gy. Palifermin increased the ED(50) to about 19 Gy in all protocols tested. Similar values were observed when chemotherapy was added before irradiation. With fractionated irradiation, palifermin increased the ED(50) for test irradiation from 5.7 +/- 1.5 Gy to 12-15 Gy, depending on the administration protocol. With chemotherapy in Week 1, two palifermin injections had no significant effect, but a third injection increased the ED(50) to 13 Gy. With chemotherapy in Week 2, all palifermin protocols resulted in ED(50) values of 13-14 Gy. CONCLUSION: A marked increase in oral mucosal radiation tolerance by palifermin was found, which was preserved in combinations with chemotherapy using cisplatin and/or 5-fluorouracil.

Effects of keratinocyte growth factor (palifermin) administration protocols on oral mucositis (mouse) induced by fractionated irradiation.

BACKGROUND AND PURPOSE: Aim of this study was to assess the impact of the administration protocol of palifermin on amelioration of oral mucositis after fractionated irradiation. MATERIALS AND METHODS: Mouse tongue ulceration was analysed as the clinically relevant endpoint. Daily fractionated irradiation (5 x 3 Gy/week, days 0 to +4, +7 to +11, with a weekend gap on days +5 and +6) was followed by graded test doses on day +14, i.e. after a second weekend gap. Palifermin (5 mg/kg) was injected subcutaneously. In the first series of experiments, the effect of three daily injections (days -3, -2 and -1) was compared with a single administration either on day -2 or -1; all animals received a further injection on day +4. In the second series, a single or three injections were given in the weekend gap between fractionated irradiation (days +5 to +6), with an additional administration on day +11. In a final protocol, single weekly injections of palifermin were given either on days -3, +4 and +11, days +4, +11 and +18, or on days -3, +4, +11 and +18. RESULTS: The ED50 (dose after which ulcer induction is expected in 50% of the mice) to single dose irradiation was 11.5+/-0.7 Gy. The ED50 for test irradiation after 10 x 3 Gy was 5.7+/-1.6 Gy. Palifermin administration before the start of fractionated irradiation and on day +4 increased the ED50 to 10-12 Gy, administration over the first weekend and on day +11 to 11-15 Gy. Administration over three consecutive weekends, starting on day -3 or day +4, increased the ED50 to 13.0+/-0.1 and 14.9+/-0.3 Gy. Single weekly KGF administrations over four weekends, including the weekend prior to and after completion of radiotherapy, showed no further increase in ED50. CONCLUSIONS: A single palifermin injection during the weekend gap before or during fractionated irradiation is as effective as three applications. Onset of the palifermin treatment during the first weekend gap between fractionated irradiation is more effective than during the weekend before radiotherapy. The effect of palifermin on oral mucositis can be increased by three weekly injections, while four injections do not yield a further increase in ED50.

Changes in the effect of dose fractionation during daily fractionated irradiation: studies in mouse oral mucosa.

PURPOSE: The aim of the present study was to quantify the fractionation effect in mouse oral mucosa during a daily fractionated protocol. METHODS AND MATERIALS: Irradiation of the snout of C3H mice was performed with 5 x 3 Gy/week. In the first experiment, graded test doses were applied to the lower tongue on Days 4, 7, 11, 14, or 18. Subsequently, a split-dose experiment was performed on the same days with 1, 2, 3, or 5 fractions (graded doses). RESULTS: The ED50 (dose expected to induce ulcer in 50% of the mice) for untreated mucosa was 10.7 +/- 1.0 Gy. Fractionated irradiation with 5 x 3 Gy/week yielded an ED50 for top-up irradiation of 6.5 +/- 1.8 Gy, 6.8 +/- 0.9 Gy, 5.3 +/- 2.1 Gy, 7.3 +/- 1.9 Gy, and 7.5 +/- 1.3 Gy on Days 4, 7, 11, 14, and 18. The ED50 values for split-dose irradiation on Day 4 increased from 6.24 +/- 1.48 Gy (1 fraction) to 9.96 +/- 1.47 Gy (5 fractions). Similarly, an increase from 4.04 +/- 1.56 Gy to 10.07 +/- 1.9 Gy was found on Day 11, and from 5.84 +/- 2.37 Gy to 9.81 +/- 2.29 Gy on Day 18. After the weekend breaks, values between 6.22 +/- 1.60 Gy (1 fraction) and 4.7 +/- 1.95 Gy (5 fractions) were observed on Day 7, and between 5.62 +/- 1.36 Gy and 5.98 +/- 2.01 Gy on Day 14. CONCLUSIONS: These results indicate that the fractionation effect in oral mucosa is consistently lost over the weekends and restored during the treatment weeks.

Clonogenic survival of human keratinocytes and rodent fibroblasts after irradiation with 25 kV x-rays.

Low energy x-rays (E(ph)

Induction of micronuclei in human fibroblasts and keratinocytes by 25 kV x-rays.

A relative biological effectiveness (RBE) not much larger than unity is usually assumed for soft x-rays (up to approximately 50 keV) that are applied in diagnostic radiology such as mammography, in conventional radiotherapy and in novel radiotherapy approaches such as x-ray phototherapy. On the other hand, there have been recent claims of an RBE of more than 3 for mammography and respective conventional x-rays. Detailed data on the RBE of soft x-rays, however, are scarce. The aim of the present study was to determine the effect of low-energy x-rays on chromosomal damage in vitro, in terms of micronucleus induction. Experiments were performed with 25 kV x-rays and a 200 kV x-ray reference source. The studies were carried out on primary human epidermal keratinocytes (HEKn), human fibroblasts (HFIB) and NIH/3T3 mouse fibroblasts. Micronucleus (MN) induction was assayed after in vitro irradiation with doses ranging from 1 to 5.2 Gy. Compared to the effect of 200 kV x-rays, 25 kV x-rays resulted in moderately increased chromosomal damage in all cell lines studied. This increase was observed for the percentage of binucleated (BN) cells with micronuclei as well as for the number of micronuclei per BN cell. Moreover, the increased number of micronuclei per micronucleated BN cell in human keratinocytes and 3T3 mouse fibroblasts suggests that soft x-rays induce a different quality of damage. For all cell lines studied the analysis of micronucleus induction by 25 kV soft x-rays compared to 200 kV x-rays resulted in an RBE value of about 1.3. This indicates a somewhat enhanced potential of soft x-rays for induction of genetic effects.

Amelioration of acute oral mucositis by keratinocyte growth factor: fractionated irradiation.

PURPOSE: The aim of the present study was to quantify the protective efficacy of recombinant human keratinocyte growth factor (rHuKGF) in oral mucosa. METHODS AND MATERIALS: Mouse tongue mucosal ulceration was analyzed as the clinically relevant end point. Fractionated irradiation of the snout with 5 daily fractions of 3 Gy was followed by graded test doses, given to a test area of the lower tongue, on Day 7. rHuKGF was injected s.c. in daily doses of 5 mg/kg before radiotherapy, during radiotherapy, over the weekend break, or a combination. Moreover, single rHuKGF injections (5 or 15 mg/kg) were given on Day -1 or on Day 4. RESULTS: In a single-dose control experiment, the ED50, i.e., the dose after which ulcer induction is expected in 50% of the mice, was 10.9 +/- 0.7 Gy. Fractionated irradiation without keratinocyte growth factor rendered an ED50 for test irradiation of 5.6 +/- 3.7 Gy. Keratinocyte growth factor increased the ED50 values to 7.8 +/- 3.3 Gy (Days -3 to -1, p = 0.01), 8.3 +/- 1.6 Gy (Days -4 to -2, p = 0.0008), 10.5 +/- 1.4 Gy (Days 0 to +2, p = 0.0002), 11.0 +/- 0.5 Gy (Days 0 to +4, p = 0.002), 10.6 +/- 1.4 Gy (Days +4 to +6, p = 0.0021), 10 +/- 0.07 (Days -3 to +1, p = 0.0001) or 11.0 +/- 0.02 (Days +4 to +8, p = 0.0001). This is equivalent to compensation of approximately 1.5 fractions of 3 Gy when rHuKGF is given before radiotherapy and 3-4 fractions in all other protocols by rHuKGF treatment. Single rHuKGF injections were similarly (5 mg/kg) or more (15 mg/kg) effective. CONCLUSIONS: In conclusion, these results indicate a marked increase in oral mucosal radiation tolerance by rHuKGF, which is most pronounced if the growth factor is applied during fractionated radiotherapy. The effect seems to be based on complex mechanisms, predominantly changes in both epithelial proliferation and differentiation processes.